Vascular ultrasound in pediatrics: utility and application of location and measurement of jugular and femoral vessels.

OBJECTIVE: To measure the depth (D p) and diameter (D m) of the internal jugular vein (IJV), femoral vein (FV), and femoral artery (FA) in pediatric patients to evaluate the clinical implications. METHODS: This study included 125 pediatric patients. All of them underwent bilateral ultrasound study of vessels and were classified into three groups based on anthropometric and demographic parameters. RESULTS: Measured mean D p values were: 0.72 (0.34) cm for the FA, 0.79 (0.35) cm for the FV, and 0.77 (0.24) cm for the IJV. Mean antero-posterior D m values were: 0.37 (0.17) cm for the FA, 0.42 (0.22) cm for the FV, and 0.59 (0.23) cm for the IJV. D p and D m increased with age (A), weight (W), height (H), and body surface area (BSA). In the lower ranges of these variables, D p was similar for all three studied vessels (0.6-0.7 cm). In the higher ranges, femoral vessel D p values (1.1-1.2 cm) were larger than jugular ones (0.9 cm). Additionally, in these low ranges, IJV D m values were larger than femoral ones (0.45-0.50 vs. 0.25 cm). In the higher ranges, diameter values were similar (0.6-0.7 cm). CONCLUSIONS: In pediatric patients, major vessels can be located and their depth and diameter measured by vascular ultrasound. In younger patients, jugular and femoral vessels had similar depth values; in older ones, they had similar diameters. Ultrasound measurements in pediatric patients could facilitate the choice of the vessel to be cannulated, the catheter diameter, and the length of the needle to be used. Vascular canalization of IJV may be recommended as the first choice because of its low depth and large diameter.

Vascular ultrasound in pediatrics: estimation of depth and diameter of jugular and femoral vessels.

OBJECTIVES: To estimate, on the basis of anthropometric and demographic variables, the depth (Dp) and diameter (Dm) of femoral and jugular vessels, which have been located and measured by ultrasound, in pediatric patients. METHOD: 750 measurements of Dp and Dm of the femoral vein (FV), femoral artery (FA) and internal jugular vein (IJV) were made in 125 pediatric patients. The values were correlated with patients' sex, weight, age, size and body surface area (BSA). RESULTS: Mean Dp values were 0.72 (0.34) cm for FA, 0.79 (0.35) cm for FV and 0.77 (0.24) cm for IJV. Mean antero-posterior Dm values were 0.37 (0.17) cm for FA, 0.42 (0.22) cm for FV and 0.59 (0.23) cm for IJV. In the studied pediatric patients, femoral and jugular vessels depth correlated with age, size, weight and BSA (R = 0.46-0.60); vascular depth could be estimated from patients' weight and size (FA-Dp: R = 0.71; FV-Dp: R = 0.72; IJV-Dp: R = 0.53). Correlation with diameter was better for FA and FV (R = 0.81-0.89) than for IJV (R = 0.42-0.51); vascular diameter could be estimated from patient's size (FA-Dm: R = 0.89; FV-Dm: R = 0.86; IJV-Dm: R = 0.52). CONCLUSIONS: FV, FA and IJV depth and diameter correlated with weight, size, age and body surface area in the studied pediatric patients. Correlation was better for femoral than for jugular vessels. Depth could be estimated from patients' weight and size, while diameter could be estimated from the size. Such estimations may facilitate the choice of vessels to be cannulated, length and diameter of cannulation needles and the diameter of catheters to be used in pediatric patients.

Celulitis orbitaria y empiema subdural como complicación de una extracción dentaria / Orbital cellulitis and subdural empyema as a complication of dental extraction

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Variation of bcl-2 expression in breast ducts and lobules in relation to plasma progesterone levels: overexpression and absence of variation in fibroadenomas.

Some women with benign breast disease eventually develop breast cancer. The mammary gland undergoes tissue remodelling according to hormonal influences, involving a balance between quiescence, proliferation, and mechanisms of cell death. Proliferation and/or apoptotic events could therefore be investigated to help understand the mechanisms of benign lesion formation and identify mastopathies with a poor prognosis. bcl-2 expression was analysed by immunohistochemistry in 75 benign mastopathies. Protein levels were quantitated with an image analyser in various epithelial structures on frozen sections, including adenoses, fibroadenomas, ductal epithelial hyperplasias, cysts, and apparently normal surrounding lobules and ducts. bcl-2 levels were equivalent in apparently normal lobules and ducts, as well as in cysts and ductal hyperplasias. bcl-2 staining was significantly higher in fibroadenomas, known to be of lobular origin [mean = 10.1, quantitative immunochemistry score (QIC) arbitrary units (AU), n = 19], than in normal lobules (mean = 5.1 AU, n = 43, P = 7 x 10(-5). bcl-2 levels in normal lobules and ducts varied according to the menstrual cycle, being higher during the follicular than the luteal phase (P = 1.8 x 10(-2) and P = 1.7 x 10(-2), respectively). This was further supported by a statistical link (P = 5 x 10(-3) between high levels of circulating progesterone and weak bcl-2 staining in lobules and ducts. This progesterone-dependent variation was absent in fibroadenomas. No statistical correlation was found between bcl-2 expression and circulating levels of oestradiol, and follicle-stimulating or luteotrophic hormones. Although these are only preliminary results, they suggest an influence of progesterone on bcl-2 expression which might be lost in fibroadenomas. A hypothesis is proposed concerning the potential involvement of altered regulation of the apoptotic process in the formation of such benign lesions.

The proto-oncogene c-fos increases the sensitivity of keratinocytes to apoptosis.

In human skin, most studies have suggested a role of c-fos or c-fos related genes in keratinocyte differentiation. The aim of our work was to more directly address this question by transfecting more or less differentiated keratinocyte cell lines (A431 and HaCaT) with constitutive expression vectors for c-Fos or c-Fos + c-Jun. Our results showed that c-Fos expression decreased keratinocyte growth, yet addition of c-Jun seemed to revert this c-Fos induced growth inhibition. Whereas no obvious differentiation program was turned on by c-Fos or c-Fos + c-Jun expression in our tissular model, apoptotic figures were observed and confirmed by in situ DNA fragmentation studies. These results do not rule out a role of c-Fos in keratinocyte differentiation but may indicate that the cell lines we used have reached an irreversible state of transformation so that they no longer respond to differentiation signals and rather die from apoptosis. These data add further evidence in favor of a role of c-Fos in epidermal homeostasis.

Expression of the erbB-2 proto-oncogene during differentiation of the mammary gland in the rat.

The erbB-2 proto-oncogene encodes a transmembrane protein (p185) that is a tyrosine kinase sharing high homology with the epidermal growth factor receptor. Its expression in mammary cell lines is modulated by estrogens, epidermal growth factor, and several other factors at the RNA and/or protein levels. We have used in situ hybridization, immunoblot, and immunohistochemistry to study the expression of erbB-2 in the rat mammary gland at various stages of differentiation. erbB-2 RNA is present at low levels in mammary glands from virgin, mid-pregnant, and lactating female rats. Increased RNA levels can be detected in early and late pregnancy. In all samples, erbB-2 RNA has been found in all epithelial cells. Immunohistochemistry with antisera directed against the intracellular domain of p185 has shown that only a minority of cells are stained in virgin and early pregnant samples, whereas no staining is seen in late pregnant and lactating mammary glands. In contrast, immunoblot analysis has detected the highest levels of p185 in late pregnancy and during lactation. This may reflect either that the cellular content of p185 is too low to be detected by immunohistochemistry, or that the epitopes are not accessible to the antisera in situ. Taken together, our data indicate that erbB-2 is expressed by mammary epithelial cells at all physiological stages and suggest that erbB-2 expression is modulated at both the RNA and protein level in vivo.

Photic regulation of c-fos gene expression in the suprachiasmatic nucleus and the circadian rhythm of photosensitivity in the mink.

The relationship between the photic stimulation of the c-fos gene product in cells of the suprachiasmatic nuclei and the photoperiodic control of testicular activity were examined in mink. Mink were kept in a short photoperiod (control, LD4:20), or in 'asymmetric skeleton photoperiods' (groups A and B). The light period for groups A and B was divided into two fractions (3 h 30 min and 30 min); the shorter fraction occurred in the night, 4 h (group A) or 8 h (group B) after the end of the main light period. There was no photic activation of the proto-oncogene c-fos on the control or group A, and 4 weeks on this photoperiodic treatment produced marked testicular development. In contrast, in group B, c-fos mRNA was induced 30 min after the end of the secondary photofraction, was maximal 30 min later and then decreased. Fos-like immunoreactivity was detected 2 h after the end of the secondary photofraction, with activity peaking 1 h later. The animals of this group remained sexually quiescent. These results suggest that photo-induction of the proto-oncogene c-fos is implicated in the gonadal inhibition induced in this species when the light period, extends into the photosensitive phase of the circadian rhythm of photosensitivity.

Cellular localisation by in situ hybridisation of cathepsin D, stromelysin 3, and urokinase plasminogen activator RNAs in breast cancer.

We have compared by RNA in situ hybridisation on serial cryo-sections the distribution of cathepsin D (cathD), stromelysin 3 (strom-3), and urokinase plasminogen activator (UPA) gene expression in different tissues of human benign and malignant mammary tumors. Cath-D expression was found to be higher in adenocarcinomas compared to non-tumoral glands. The cath-D RNA was located in mammary epithelial cancer cells rather than in fibroblasts, indicating that the cath-D gene was overexpressed in cancer cells, where the corresponding protein determined by immunohistochemical staining had been shown to be accumulated (E Roger et al., Human Pathol 25: 863-871,1994). In contrast strom-3 RNA in adjacent tissue sections used as a control of tissue localisation was mostly expressed in peritumoral fibroblasts rather than in cancer cells confirming previous results of Basset et al. and validating our methodology. UPA RNA was detected both in tumor cells and in stromal cells. In benign lesions the 3 protease RNAs were mostly found in epithelial cells. Stromal cells expressed UPA RNA in 5 of 7 lesions, cath-D and strom-3 in only one sample. We conclude that in breast cancer patients, cath-D gene expression is increased in epithelial mammary cancer cells at the RNA level as well as at the protein level, suggesting an altered transcriptional regulation. In non malignant lesions, the distribution was different with a predominant distribution in epithelial mammary cells for the 3 protease messenger RNA.

In vivo stimulation by tamoxifen of cathepsin D RNA level in breast cancer.

We have previously shown that 3 weeks of treatment with tamoxifen, of patients with primary breast carcinomas, increased cytosolic cathepsin D protein in oestrogen receptor (ER) positive tumours [Maudelonde et al., Cancer 1989, 63, 1265-1270]. In order to investigate the mechanism of this increase and to eliminate a transient flare-up effect, we semi-quantified cathepsin D RNA levels by in situ hybridisation in 32 breast carcinomas from patients treated with tamoxifen for 3 weeks prior to surgery and in 35 breast cancer patients receiving no tamoxifen. We found that tamoxifen increased cathepsin D RNA level regardless of the ER status of the tumours. In ER positive tumours, tamoxifen increased the cathepsin D RNA level to the same extent as cytosolic cathepsin D protein but not in ER negative tumours. The induction of cathepsin D RNA by tamoxifen in ER positive tumours was probably due to its agonist activity, also observed in vitro in breast cancer cell lines. These results suggest that the cathepsin D gene is inducible by oestrogens in ER positive breast cancer as it is in breast cancer cell lines.

Correlation between mannose-6-phosphate/IGFII receptor and cathepsin D RNA levels by in situ hybridization in benign and malignant mammary tumors.

We evaluated levels of mannose-6-phosphate/insulin growth factor-II receptor (M6P/IGFII-R) RNA in 37 breast cancer tumors by quantitative in situ hybridization using a computer-aided image analyzer and compared them to cathepsin D RNA and protein levels in the same tissues. Breast cancer cells expressed more cathepsin D and M6P/IGFII-R RNA than fibroblasts in the same tumors. We found a significant increase of cathepsin D RNA (P = 1 x 10(-5)) and M6P/IGFII-R RNA (P = 0.02) in breast cancer cells compared to epithelial cells of benign mastopathies. There was a positive correlation (r = 0.65; P = 1 x 10(-5)) between M6P/IGFII-R and cathepsin D RNA levels measured on serial sections. This contrasted with the inverse relationship of these 2 RNA species in breast cancer cell lines where estrogen down-regulates M6P/IGFII receptor RNA levels. Moreover, in vivo we found no correlation between the M6P/IGFII-R RNA level and menopausal or estrogen receptor status, suggesting that the in vivo regulation of M6P/IGFII-R RNA differs from its in vitro regulation in cell lines. The M6P/IGFII-R RNA level was not correlated with cathepsin D status, histological grade, and tumor size but was significantly higher in lymph node-positive tumors (P = 0.047). The M6P/IGFII-R could therefore be an additional parameter to predict aggressive breast cancers, complementing cathepsin D assays and other more classical prognostic parameters.

Potential value of increased MYC but not ERBB2 RNA levels as a marker of high-risk mastopathies.

MYC and ERBB2 levels were measured in 38 benign breast diseases using a semiquantitative in situ hybridization technique. Mean levels of MYC and ERBB2 gene expression in benign tissues were similar to those measured in 15 breast cancers with no amplification at the loci concerned. Interestingly, MYC but not ERBB2 RNA levels were increased (t-test, P = 0.03) in benign mastopathies of patients with a first-degree (mother/sister) family history (FH) of breast cancer. Among patients without a first-degree FH, MYC RNA levels were significantly higher (t-test, P = 0.02) during the follicular (preovulatory) than the luteal (post-ovulatory) phase and also significantly higher than levels observed in patients with no menstrual cycle (peri- or postmenopausal) (P = 0.004), indicating an in vivo hormonal regulation of MYC. After exclusion of the first-degree FH patients a higher MYC expression was detected in atypia than in other histological types at the follicular but not at the luteal phase, suggesting an increased sensitivity of these high-risk lesions to estrogens. We propose that in addition to a family history and proliferative atypia, elevated MYC RNA levels during the post-ovulatory phase could potentially be used as a marker of the risk of developing breast cancer. The increase in MYC RNA in high-risk breast diseases also suggests that MYC deregulation might be involved in the early stages of mammary carcinogenesis.

C-fos and c-jun proto-oncogene expression is decreased in psoriasis: an in situ quantitative analysis.

Psoriasis is a common, sometimes severe, non-malignant skin disease characterized by hyperproliferation and abnormal differentiation of keratinocytes. Because proto-oncogenes are implicated in both cell proliferation and differentiation, their expression could be modified in skin diseases such as psoriasis. The c-fos and c-jun proto-oncogenes, whose products associate to form a heterodimeric transcription factor, are among the first genes to be expressed when certain cells are stimulated to either proliferate or differentiate. Recent studies in our laboratory have shown that the c-fos proto-oncogene is highly expressed in normal human adult skin. In the present study, we used in situ hybridization with RNA to compare the expression and localization of c-fos and c-jun transcripts in 15 lesional and non-lesional psoriatic skin samples. Two clinical variants of psoriasis were studied: the most severe and chronic form or plaque-type psoriasis (N = 10) and rapidly resolutive guttate-type psoriasis (N = 5). Quantitative analysis was performed using a semi-automatic image analyzer and the "Starwise grain" software program. Our control samples included 10 normal skins and eight specimens from other benign hyperproliferative non-psoriatic skin diseases, consisting of three with inflammation (seborrheic dermatitis and atopic dermatitis), and 5 without inflammation (seborrheic keratoses). Control genes we used for in situ hybridization and RNA integrity were keratin 14, which is expressed in the epidermis and was normally expressed in all tissue analyzed, and ribosomal RNA. Our data showed that c-fos and c-jun were expressed to an equivalent extent, both spatially and quantitatively, in all specimens tested. Expression was significantly decreased in plaque-type but not in guttate-type psoriasis. It was also decreased in the three other benign inflammatory cutaneous hyperproliferative disorders, but not in the five non-inflammatory cases. These results were surprising because hyperproliferation was here associated with a decrease in proto-oncogene expression, thus suggesting that c-fos and c-jun do not play a crucial role in the control of keratinocyte proliferation in vivo. However, their reduced expression in some abnormally differentiated skins indicates that both c-fos and c-jun proto-oncogenes may play a key role in keratinocyte differentiation. Their altered expression correlated with severity of the disease and the presence of an inflammatory infiltrate. These data offer a new insight into the role and regulation of these proto-oncogenes in vivo in humans.

Computer-aided quantification of RNA levels detected by in situ hybridization of tissue sections.

A semi-automatic program, designed for non-computer scientists, was developed for quantification of RNA levels detected by in situ hybridization in heterogeneous tissues. A video camera was used to acquire microscopic images of autoradiographed tissue sections which are then digitized on a video monitor for semi-automated quantification of silver grains. We describe a data entry and analysis procedure for systematic quantification of RNA levels in which about 300 cells per tissue sample can be analysed within 10 min. When compared with visual counting, computer-aided quantification was found to be more objective and reliable, with the highest variation coefficient between individuals being 7.5% using computer-aided quantification, compared to 24% with manual counting of the same section areas. A comparative study of c-myc oncogene expression in 11 mammary adenocarcinomas from 3 independent experiments showed the good reproducibility of results using the computer-aided method, with an 18% maximum variation between experiments. The program, with its simple user-interface, reliability and rapidity, is convenient for measuring specific genetic expression levels in clinical studies requiring large numbers of specimens.

Decrease of c-erbB-2 and c-myc RNA levels in tamoxifen-treated breast cancer.

The c-myc, c-erbB-2, hst and int-2 oncogenes are frequently amplified and/or overexpressed in human breast carcinomas. We studied the effect of tamoxifen on RNA levels of these oncogenes in 19 breast cancer patients treated for 3 weeks prior to surgery as compared with 22 control patients. RNA levels were measured by in situ hybridization coupled with computer-aided quantification. c-myc and c-erbB-2 expression was high in the control population (mean values: 23.4 and 29.1 grains/cell respectively) and significantly decreased in the tamoxifen-treated population (mean values: 14.6 and 7.4 grains/cell respectively) (P = 0.018, P = 0.003 respectively); hst and int-2 RNA levels were low (2-6 grains/cell) and not significantly altered by the treatment. There was a correlation between gene amplification and expression for c-erbB-2 (P = 0.0005) and hst (P = 0.02) in the control population. Elevated c-erbB-2 RNA level was correlated with the absence of estrogen (P = 0.02) or progesterone (P = 0.05) receptors. In the ER+ population, the tamoxifen-treated group had significantly lower c-myc expression levels than the control group (P = 0.04) which is in agreement with the estrogen induction of c-myc in ER+ T47D cell line and its inhibition by antiestrogens. Surprisingly, c-erbB-2 expression in the tamoxifen-treated group was significantly diminished in the ER- (P = 0.02) and PR- (P = 0.01) populations. This effect was not observed in the ER- BT474 cell line. These results suggest that in vivo tamoxifen decreases c-myc and c-erbB-2 RNA levels in breast cancer cells via two different mechanisms. To our knowledge this is the first evidence of in vivo down regulation of a gene by tamoxifen in ER- breast cancer cells.

Regulation of fatty acid synthetase ribonucleic acid in the human endometrium during the menstrual cycle.

Fatty acid synthetase (FAS) is induced by progesterone in MCF7 and T47D breast cancer cell lines. We studied a possible in vivo regulation of expression of this gene by looking for FAS RNA in human endometrial biopsies at various periods of the menstrual cycle, using a cloned cDNA FAS probe. By Northern blot analysis, we detected the 8-kilobase FAS RNA throughout the cycle in 7 uterine samples. RNA in situ hybridization analysis of frozen sections from 22 endometrial biopsies showed that FAS RNA was present during follicular and luteal phases of the menstrual cycle in stromal and epithelial cells. RNA levels were quantified by counting autoradiographic silver grains using a computer-aided image analyzer. FAS RNA levels were significantly higher in epithelial cells than in fibroblasts (P less than 2 x 10(-5]. Furthermore, in both cell types, mean FAS RNA concentrations were higher in biopsies removed during the luteal phase than the follicular phase of the menstrual cycle (P = 2 x 10(-3) and 9 x 10(-5), respectively). A 2- to 3-fold increase in FAS RNA levels between days 8-14 and days 22-24 was detected in 2 normal patients who had previously undergone 2 successive biopsies. This increase was not observed in 2 patients with low plasma estradiol and progesterone concentrations, indicating a probable dysovulation. We conclude that FAS normally increases in both stromal and epithelial endometrial cells during the luteal phase. This increase is probably due to progesterone, which implies that FAS is induced in normal endometrium, as demonstrated in breast cancer.

Expression of the progestin-induced fatty acid synthetase in benign mastopathies and breast cancer as measured by RNA in situ hybridization.

We have shown previously that fatty acid synthetase (FAS) is specifically induced by progestins in human breast cancer cell lines. To test the potential value of FAS as a clinical marker in breast diseases, we measured FAS expression in frozen sections of 22 benign and 27 malignant mammary tumors using in situ hybridization with the [35S]UTP alpha S-labeled FAS anti-sense mRNA. The hybridized RNA was quantified with an IMSTAR computerized image analyzer. We found FAS RNA in epithelial cells, but no labeling was detected in the connective tissue. In breast cancer, we found no correlation between FAS expression and estrogen receptor and progesterone receptor concentrations or status. However, the level of FAS was significantly (P less than .02) higher in premenopausal than in post-menopausal patients and increased with the grade of tumor differentiation (P less than .005 between the poorly and well-differentiated tumors). In benign mastopathies, high levels of FAS RNA were found in some cysts (mostly with apocrine metaplasia). In lobules, the FAS RNA level increased proportionally to the degree of proliferation determined by histological examination (P less than .015) and correlated with the H4 histone level measured in an adjacent section using in situ hybridization (r = 0.85, P less than .001). In ductal structures, a lower correlation (r = 0.64, P less than .01) was found between FAS and H4 RNA levels. We conclude that FAS RNA is overexpressed in some mammary tumors and may be useful in predicting high-risk mastopathies and less aggressive breast cancers.

High levels of c-fos proto-oncogene expression in normal human adult skin.

The proto-oncogene c-fos is thought to play an important role in the modulation of cell growth and differentiation. In normal tissues that have been studied to date, c-fos expression has been found to be regulated in a tissue-specific manner. Actually, little is known about its expression in normal human adult skin (NHAS). Moreover, the epidermis is a useful tissue to study the role of cellular oncogenes because keratinocytes can be observed simultaneously in their proliferative as well as differentiated state. We studied c-fos expression in NHAS using different molecular approaches which permit us to characterize and localize c-fos products within the epidermis, specifically, at the RNA level by Northern blot and in situ hybridization, and at the protein level by immunofluorescence and Western blotting. Here, we show that both c-fos mRNA and protein are present at high levels in NHAS. These results contrast with the low level of c-fos expression reported for most human adult tissues. Furthermore, c-fos expression is visible throughout the epidermal layers indicating that it is not restricted to proliferating basal cells. The epidermis, therefore, represents the first human adult tissue where c-fos is expressed at high levels in vivo and provides an interesting model to further elucidate the role of this proto-oncogene in normal and pathologic conditions.