Detection of cervical intraepithelial neoplasia in vivo using confocal endomicroscopy.

OBJECTIVE: A high resolution optical imaging device may offer a clinically useful adjunct to colposcopy for the diagnosis and assessment of cervical precancer. This study describes the clinical evaluation of a miniaturised confocal endomicroscope for the quantitative and qualitative assessment of cervical intraepithelial neoplasia (CIN) in vivo. DESIGN: A descriptive study (n = 25) was performed to assess the usability of confocal endomicroscopy to image the cervix. A prospective study (n = 15) then evaluated the diagnostic accuracy of the technique. SETTING AND POPULATION: Patients undergoing colposcopy for treatment of CIN1-CIN3 were examined using confocal endomicroscopy. METHODS: A 5% solution of acetic acid was used to enhance the colposcopic features of the atypical region. Normal and abnormal regions of the cervix were then imaged following topical application of a fluorescent dye (acriflavine). MAIN OUTCOME MEASURES: Confocal images were analysed to develop a scoring system to grade different levels of CIN. Microscopic features were correlated with histology from biopsy. RESULTS: Confocal endomicroscopy enabled microscopic imaging of cellular and subcellular structures in vivo at colposcopy. Imaging at increasing depth showed morphological features including dermal papillae, endocervical glands and the squamo-columnar junction. CIN was characterised by an increase in nuclear density, size and cellular atypia. The sensitivity for detection of CIN was 97%. The specificity for predicting the grade of abnormality was 80% for normal-CIN1 and 93% for CIN2-CIN3. CONCLUSIONS: Confocal endomicroscopy is a sensitive imaging tool for detection and assessment of CIN. The technique enables in vivo imaging of cervical histology and the potential for 'see-and-treat' workflows.

Haplotype-based search for SNPs associated with differential type 1 diabetes risk among chromosomes carrying a specific HLA DRB1-DQA1-DQB1 haplotype.

AIM: The aim of this study was to test chromosomes carrying the same DRB1-DQA1-DQB1 haplotype for single nucleotide polymorphisms (SNPs) in the major histocompatibility complex (MHC) that might mark subgroups of the haplotype with different risks for type 1 diabetes (T1D). METHODS: Chromosomes from T1D children, their parents and non-diabetic siblings in families of the Type 1 Diabetes Genetics Consortium (T1DGC) were analysed by two haplotype-based methods: (i) logistic regression analysis restricted to phased chromosomes carrying the same DRB1-DQA1-DQB1 haplotype but differentiated by the two alleles at MHC SNPs, which were individually tested for association with T1D and (ii) homozygous parent transmission disequilibrium test (TDT) for biased transmission of a SNP allele to diabetic children from parents who are heterozygous at the SNP but homozygous for the specific DRB1-DQA1-DQB1 haplotype being evaluated. RESULTS: A number of SNPs gave nominally significant (p < 0.05) evidence of marking two subsets of the 301-501-201 haplotype that might differ with respect to their diabetogenic potency. However, none of the SNPs achieved experiment-wide significance and hence may be false-positive associations. CONCLUSIONS: We discuss limitations and possible deficiencies of our study suggesting further work that might yield more robust SNP associations marking two subgroups of a DRB1-DQA1-DQB1 haplotype with different T1D risks.

In vivo detection of experimental ulcerative colitis in rats using fiberoptic confocal imaging (FOCI).

Fiberoptic confocal imaging (FOCI) is a noninvasive microscopic technique that enables subsurface imaging of living tissue in vivo. The aim of the present study was to assess the suitability of FOCI for the in vivo detection of early subsurface changes in the mucosal architecture of the colon in a rat model of ulcerative colitis. Mild colitis was induced in Sprague-Dawley rats (180-250 g) by the oral ingestion of 5% (w/v) dextran sulfate sodium (DSS; Mr 40,000 Da) in drinking water. Control animals were provided with water ad libitum. After three, five or seven days of oral consumption of DSS, the mucosal surface of the colon of anesthetised rats was surgically exposed. Morphological changes in the mucosa were examined (Optiscan F900e personal confocal system with rigid endomicroscope attachment; excitation 488 nm argon ion laser, detection above 515 nm) following the topical application of a fluorescent dye (fluorescein, eosin, or acridine orange). Confocal images were correlated with conventional histology and clinical parameters including occult blood and stool consistency. Histological evaluation of colon sections demonstrated that DSS-induced colitis was characterized by focal loss of mucous crypts, loss of epithelial cells, and neutrophilic infiltration into the mucosa. The extent of mucosal damage was positively correlated with the time of ingestion of DSS. Morphological changes associated with disease activity could be detected microscopically in vivo using FOCI but were not evident by visual inspection of the colon surface. Acridine orange enabled imaging of the colonic crypts at the surface of the mucosa. Morphological changes associated with colitis, including inflammatory cell infiltrate, crypt loss, and crypt distortion, could be detected using this fluorophore. Application of fluorescein and eosin enabled subsurface imaging of the lamina propria surrounding the crypts; however, no change in structure was detected in association with colitic disease activity. This study has shown that the topical application of acridine orange enables in vivo imaging of early colitis in a rat model. FOCI may be suitable for the diagnosis and monitoring of human inflammatory bowel disease.

Autofluorescence of skin burns detected by fiber-optic confocal imaging: evidence that cool water treatment limits progressive thermal damage in anesthetized hairless mice.

BACKGROUND: Although full-thickness burns present no difficulty to clinical judgment, accurate assessment of burn depth immediately after injury in partial thickness burns has always been difficult. METHODS: Thermal burns (applied by a 3-mm-diameter brass rod heated to 50 degrees--80 degrees C for 20 seconds) were induced on the skin of anesthetized hairless mice. Anesthesia was maintained throughout all experiments. Both burns and normal skin were investigated noninvasively in vivo using fiber-optic confocal imaging (FOCI) microscopy (excitation, 488 nm; detection, 505 nm). RESULTS: Autofluorescence was detected in burned skin, and the depth of the autofluorescent region was found to correlate with the intensity of heat applied. Cool water treatment (for 20 minutes immediately after burn induction) significantly reduced the progressive increase in autofluorescence in deeper layers of the skin over the 4-hour postburn observation period. Histology showed burn-associated changes at a lower temperature than that at which autofluorescence was first detected in vivo by FOCI. However, there was a good correlation (r = 0.78) between depth of damage revealed by FOCI compared with that by histology. CONCLUSION: These results suggest that FOCI may be used to provide an index of burn depth.

In vivo detection of small subsurface melanomas in athymic mice using noninvasive fiber optic confocal imaging.

Fiber optic confocal imaging, following intravenous administration of fluorescently labeled antibodies and Texas Red-dextran, enabled in vivo detection of melanoma and surrounding blood vessels in athymic mice. Human melanoma cells (three cell lines) and cultured normal human skin cells were implanted intradermally into the haunch skin of anesthetized athymic BALB/C mice and allowed to grow to a maximum size of 2 mm diameter. Using three different fluorescein-isothiocyanate-labeled antimelanoma antibodies, single channel confocal images of melanoma cells were obtained in vivo. Using noninvasive techniques, the overall in vivo melanoma detection rate for tumors within 0.2 mm of the skin surface was 84% (27 of 32 tumors). Normal cultured human skin cells were found to have little or no fluorescence after administration of the fluorescein-isothiocyanate-labeled antibodies and tumors were not labeled by an isotype control antibody. Dual channel imaging of the implanted melanoma tumor and surrounding dermal vasculature in vivo showed increased blood vessel density at the melanoma site. Conventional immunoperoxidase histology confirmed that fiber optic confocal imaging was able to detect melanoma tumors up to 0.2 mm below the skin surface, in vivo.

Immunohistochemical localization of prostaglandin G/H synthase 1 and 2 in sheep placenta after glucocorticoid-induced and spontaneous labour.

Enhanced prostaglandin production and release by the placenta is an essential element in the normal transition to labour in many animal species. In sheep, expression of prostaglandin G/H synthase (PGHS) is the central enzyme regulating this process. In this study immunohistochemistry was used to examine the distribution of cells expressing PGHS-1 and PGHS-2 in ovine placenta in association with spontaneous parturition (n = 6) and glucocorticoid-induced labour (n = 5). Labour was induced in ewes after the intrafetal injection of betamethasone on day 131 of gestation. Animals administered an intrafetal injection of isotonic saline (n = 5) acted as non-labour controls. In placentomes collected from all groups, immunoreactive PGHS-1 was present in the mononuclear trophoblast cells of the fetal placenta. Cells in the maternal mesenchyme and epithelial syncytium were weakly immunopositive for this enzyme. PGHS-1 immunoreactivity was also demonstrated in the endothelial cells of the chorionic vessels. The PGHS-2 isozyme was localized exclusively to the trophoblast epithelial cells. Immunoreactive PGHS-2 was not detectable in the maternal epithelial syncytium or the stroma of the cotyledons. The binucleate cells of the fetal placenta were consistently immunonegative for both PGHS isozymes. These results indicate that the cellular localization of PGHS-1 and PGHS-2 in ovine placenta does not change during the last 15 days of pregnancy. Co-localization of these isozymes indicates that the source of arachidonic acid and the site of prostanoid formation are the same. Quantitation of the percentage area of positive staining for PGHS-1 and PGHS-2 using image analysis software demonstrated a significant increase in PGHS-2 in the fetal trophoblast after glucocorticoid-induced labour and spontaneous parturition. This finding indicates that increased formation of the PGHS-2 isozyme is responsible for the large increase in prostaglandin production by the ovine placenta at term labour.

Localisation and temporal changes in prostaglandin G/H synthase-1 and -2 content in ovine intrauterine tissues in relation to glucocorticoid-induced and spontaneous labour.

Parturition in the ewe is preceded by an increase in the synthesis of prostaglandins (PGs) by gestational tissues. To establish the uterine source of these PGs, placental cotyledons, fetal membranes and maternal uterine tissues were collected from ewes (n=6) at spontaneous parturition. Solubilised tissue extracts were prepared and analysed by Western blots using polyclonal antibodies to PG G/H synthase-1 and -2 (PGHS-1 and PGHS-2). PGHS-1 was expressed by all intrauterine tissues at term labour. Densitometric analysis of Western blot autoradiographs showed that the fetal membranes and maternal cervix contained the largest amounts of PGHS-1. PGHS-1 enzyme content of ovine amnion was significantly greater than that of either chorion or allantois (P<0.05). PGHS-1 protein content of myometrial, endometrial and cotyledonary tissue extracts was minimal. Formation of the PGHS-2 isozyme was confined to placental tissue at term labour. PGHS-2 protein levels in sheep placenta were significantly higher than those of PGHS-1 in all intrauterine tissues examined. This result supports the hypothesis that PGHS-2 is a major contributor to PG formation at term labour. To elucidate the developmental changes in PGHS-1 and PGHS-2 relative to labour onset, an experimental paradigm of glucocorticoid-induced delivery was used. Previous characterisation and validation of this labour model demonstrated that direct, transabdominal, intrafetal injection of the synthetic glucocorticoid betamethasone (5.7 mg in 1 ml aqueous vehicle) on day 131 of gestation induced labour onset in 56.6+/-0.8 h (mean+/-s.e.m.). As the latent period to induced-labour was known, the time course of enzyme formation could be ascertained. Sheep (n=20) were killed by barbiturate injection at various time intervals post-injection (0, 14, 28, 42 and 56 h). Tissue extracts collected at post-mortem examination were prepared and analysed by Western blots. PGHS-2 was induced in ovine cotyledon in a time-dependent fashion following glucocorticoid injection (P<0.05). There was a 12-fold increase in abundance between the time of betamethasone administration (0 h) and established labour (56 h). The PGHS-2 isozyme was not detected in any of the other tissues examined. In contrast, formation of the PGHS-1 isozyme did not change in relation to induced-labour in any of the intrauterine tissues. This finding is consistent with constitutive formation of PGHS-1. Previous studies have demonstrated a rise in PG production in association with glucocorticoid-induced labour and spontaneous delivery. The results of the present study indicate that this rise in PG production is due to increased formation of the PGHS-2 isozyme in ovine cotyledon. PGHS-2 appears to be induced by exogenous glucocorticoid administration and/or the mechanisms controlling ovine parturition. The role of PG formation by the fetal membranes is yet to be elucidated.

Expression of prostaglandin G/H synthase-1 and -2 in ovine amnion and placenta following glucocorticoid-induced labour onset.

Parturition in the sheep is preceded by an increase in the synthesis of prostaglandins by intrauterine tissues. Prostaglandin G/H synthase (PGHS) is the central enzyme involved in prostanoid production. Its expression is enhanced during late gestation in the ewe. Recent studies have identified two PGHS isozymes, termed PGHS-1 and PGHS-2. The labour-associated expression of the two isozymes of PGHS in the sheep has not been characterized. This study investigated the changes in expression of immunoreactive PGHS-1 and PGHS-2 in ovine amnion and placenta following glucocorticoid-induced labour. Ewes underwent surgery to implant fetal and maternal vascular cannulae and uterine electromyogram electrodes between 118 and 125 days of gestation. Fetal sheep were administered either the glucocorticoid betamethasone (n = 5) or saline (control n = 6) by direct transabdominal intrafetal injection. Ewes from the betamethasone-injected group were killed in the first stage of labour as indicated by uterine electromyographic activity. Ewes from the saline-injected group were killed at the same time to obtain age-matched control tissue. The time taken to euthanasia following induced-labour onset in the glucocorticoid-injected animals was 56.6 +/- 0.8 h post-injection. Plasma endocrine profiles in the maternal and fetal circulation following glucocorticoid injection were comparable to those observed following normal spontaneous delivery. At post-mortem, amnion and cotyledons were collected in liquid N2 and stored at -70 degrees C. Solubilized tissue extracts were prepared and analysed by Western blots using polyclonal antibodies to PGHS-1 and PGHS-2 isozymes. Fetal amnion contained PGHS-1 isozyme at day 133 of gestation, as demonstrated in the saline-injected animals. Slightly higher PGHS-1 immunoreactivity was observed following induced-labour onset, although this did not reach statistical significance (P > 0.05). PGHS-2 enzyme was not detectable in amnion. PGHS-2 expression was also not induced following labour onset. In contrast, PGHS-2 demonstrated enhanced expression following glucocorticoid-induced labour in ovine cotyledon. This tissue contained PGHS-1 enzyme, but immunoreactive levels were minimal and demonstrated limited regulation at labour. These data suggest that the previously reported rise in placental PG production at term in the sheep is predominantly due to increased expression of the PGHS-2 isozyme. This suggests that PGHS-2 contributes to PG production at term labour in sheep or is induced by the mechanisms controlling ovine parturition. PGHS-1 isozyme is produced constitutively in ovine amnion and may contribute to the gestational increase in PG formation by intrauterine tissues.

Klinefelter's syndrome in adolescence.

Twelve boys with Klinefelter's syndrome (47,XXY) identified by sex chromatin screening at birth were examined at between ages 16 and 18 years, together with 12 controls matched for social class and birth order from the same newborn population. Physical examination, psychometric assessment, personality, and degree of psychosexual development were assessed without knowledge of the karyotype. Anthropometry showed increased leg length and decreased head circumference in the XXY boys. Gynaecomastia was present in 4 boys, and testicular volume was reduced in the majority but one boy had normal sized testes. On the Wechsler intelligence scale there was a significant reduction in verbal score but not in either performance or full-scale score compared with the controls. Although appreciable differences were found in growth, personality, intelligence test scores, and psychosexual development, these were of small degree.