Upregulation of CD44 expression by interleukins 1, 4, and 13, transforming growth factor-beta1, estrogen, and progestogen in human cervical adenocarcinoma cell lines.

Although cervical adenocarcinoma constitutes approximately 10-20% of primary malignant tumors of the uterine cervix, its pathogenesis is less well understood than that of the corresponding squamous cancer. CD44 is a cell surface glycoprotein postulated to play a role in many biologic processes including tumor growth and metastasis. We have previously reported from immunohistochemical studies that a particular CD44 variant (CD44v5) is consistently overexpressed in endocervical neoplasia. It thus has potential as a diagnostic marker and even as a target for therapeutic approaches directed against specific epitopes. The aim of this study was to investigate which cytokines and hormones are capable of modulating CD44v5 expression, using a cell culture model. The effects of interleukin (IL)-1alpha, IL-1beta, IL-4, IL-13, transforming growth factor (TGF)-beta1, estrogen, and progestogen on CD44v5 expression were examined in cultures of three human cervical adenocarcinoma cell lines (HeLa, HeLa229, and HS588T). Expression was assessed using dual fluorescence-labeled flow cytometry and western blotting techniques. It was found that incubation of cultures for 72 h with IL-1alpha, IL-1beta, IL-4, IL-13, TGF-beta1 (all at 0.1-10 ng/mL), estrogen (5-10 ng/mL), or progestogen (5-20 ng/mL) induced significant upregulation of CD44v5. These factors are likely to exert a similar stimulatory influence in vivo and may contribute to the process of carcinogenesis.

A study of the morphological parameters of cervical squamous epithelium.

Electrical impedance spectroscopy is a technique that has been investigated as a potential method for the diagnosis of epithelial carcinomas. Finite element modelling can provide an insight into the patterns of current flow in normal and pathological epithelium and hence aid in the process of probe design optimization. In order to develop a finite element model of the structure of normal and precancerous cervical squamous epithelium, it was first necessary to obtain the mean values and ranges of a number of morphological tissue parameters. The most important parameters in discriminating normal from neoplastic tissue were identified as being cell size and shape distribution, nuclear-to-cytoplasmic volume ratio and volume of extracellular space. A survey of the literature revealed an absence of reliable quantitative data for these parameters. We therefore present the results of our own basic image analysis on normal and pathological tissue sections, which we hope will be of use to other workers wishing to model cervical squamous epithelium, or other similar tissue structures.

Relation between tissue structure and imposed electrical current flow in cervical neoplasia.

BACKGROUND: When an electrical potential is applied to human tissue, the pattern of the resulting current flow is determined by the shapes, arrangements, and internal structure of the tissue cells. By measurement of the electrical current patterns over a range of frequencies, and use of an inverse modelling procedure, electrical variables describing the tissue structure can be calculated. We used this method to develop a screening technique for the detection of cervical precancers. METHODS: We used a pencil probe (diameter 5 mm) to measure electrical impedance spectra from eight points on the cervix in 124 women with abnormal cervical smears. Variables that should be sensitive to the expected tissue changes were calculated. These were compared with the colposcopic results. FINDINGS: The measured electrical impedance changes were those predicted on the basis of the expected tissue structures. Measurements made on normal squamous tissues were well separated from those made on precancerous tissues. We constructed receiver-operating-characteristic curves, comparing measurements made on normal tissue and that showing cervical intraepithelial neoplasia grade 2/3; the area under the curve was 0.951. These groups of women could be separated with a sensitivity of 0.92 and a specificity of 0.92. INTERPRETATION: Characteristics of the electrical impedance spectra of tissues can be explained by changes in cell arrangements (layering) and in the size of the nuclei. This relation opens the way to deriving tissue structure from electrical impedance spectral measurements. We show that this approach can be used to give good separation of normal and precancerous cervical tissues.

CD44 is a marker of endocervical neoplasia.

The expression of CD44s, CD44v4, CD44v5, CD44v7-8, and CD44v10 was investigated immunohistochemically in a variety of neoplastic cervical lesions. Normal endocervical columnar cells exhibited no reactivity for any of the antibodies, whereas the subcolumnar reserve cells were strongly positive for CD44s, CD44v5, and CD44v7-8. In some cases, positive cells were identified in the stroma surrounding the endocervical glands and adjacent to reserve cells. Cervical glandular intraepithelial neoplasia and adenocarcinoma showed consistent immunoreactivity for CD44v5. There was no significant change in CD44 immunoreactivity in squamous cell carcinoma compared with normal epithelia and cervical intraepithelial neoplasia. These findings lend support to the origin of carcinoma of the cervix from a common progenitor reserve cell and suggest the origin of reserve cells from the stroma. CD44v5 may be useful as a diagnostic marker of endocervical neoplasia and could provide a target for therapeutic approaches directed against specific epitopes.

Conclusions and recommendations from the Helene Harris Memorial Trust Fifth Biennial International Forum on Ovarian Cancer, May 4-7, 1995, Glasgow, UK.

The Fifth Biennial International Forum on Ovarian Cancer was held by the Helene Harris Memorial Trust in Glasgow, UK. The main points of the presentations given by the invited speakers, together with the fruits of extensive discussions are presented here as a series of conclusions and recommendations which should be given consideration by all those having an interest in researching or treating ovarian cancer. The individual points are grouped into topics considering whether there is an identifiable ovarian pro-cancer, whether ovarian cancer is preventable, advances in familial ovarian cancer, its molecular genetics, ways of optimizing treatment, obstacles to successful treatment and approaches to gene therapy.

Continuous extradural infusion of lignocaine 0.75% vs bupivacaine 0.125% in primiparae: quality of analgesia and influence on labour.

We studied 86 primiparous women with uncomplicated pregnancy and labour requesting extradural analgesia in labour. All the women were over 36 weeks of gestation with a cephalic-presenting singleton fetus. The women were allocated randomly to two groups: group A, who received an extradural infusion of lignocaine 0.75%, after an initial dose of 10 ml of lignocaine 1.5%, and group B, who received an infusion of bupivacaine 0.125% after an initial dose of 10 ml of bupivacaine 0.25%. All the women had their labour actively managed. Assessment of analgesia during labour and delivery, and the requirements for additional top-ups were noted, as were mode of delivery, requirement for oxytocic augmentation and incidence of fetal distress. Maternal and umbilical cord plasma concentrations of lignocaine were measured at delivery in 12 women receiving extradural lignocaine. There were no statistically significant differences between the two groups in terms of the mode of delivery, incidence of fetal distress, fetal heart rate abnormalities, or Apgar scores of the babies. Women in the bupivacaine group had a significantly better quality of analgesia during both the first and second stages of labour (p = 0.0005) and required fewer top-ups than those in the lignocaine group. However, the requirement for oxytocin augmentation during the first and second stages of labour was significantly less in the lignocaine group (p = 0.004). Similarly, the duration of the second stage was shorter compared with the bupivacaine group. In spite of high plasma concentrations of lignocaine, no side effects were noted in either mothers or babies.

Quantitation of radiation-, chemical-, or enzyme-induced single strand breaks in nonradioactive DNA by alkaline gel electrophoresis: application to pyrimidine dimers.

We have developed an alkaline agarose gel method for quantitating single strand breaks in nanogram quantities of nonradioactive DNA. After electrophoresis together with molecular length standards, the DNA is neutralized, stained with ethidium bromide, photographed, and the density profiles recorded with a computer controlled scanner. The median lengths, number average molecular lengths, and length average molecular lengths of the DNAs can be computed by using the mobilities of the molecular length standards. The frequency of single strand breaks can then be determined by comparison of the corresponding average molecular lengths of DNAs treated and not treated with single strand break-inducing agents (radiation, chemicals, or lesion-specific endonuclease). Single strand break yields (induced at pyrimidine dimer sites in uv-irradiated human fibroblasts DNA by the dimer-specific endonuclease from Micrococcus luteus) from our method agree with values obtained for the same DNAs from alkaline sucrose gradient analysis. The method has been used to determine pyrimidine dimer yields in DNA from biopsies of human skin irradiated in situ. It will be especially useful in determining the frequency of single strand breaks (or lesions convertible to single strand breaks by specific cleaving reagents or enzymes) in small quantities of DNA from cells or tissues not amenable to radioactive labeling.

Spectrofluorimetric analysis and buccal absorption of medifoxamine.

Medifoxamine is a new investigational antidepressant drug. Its buccal absorption at pH 5-9, which can be considered as an in-vivo model of passive drug transfer through a lipid membrane, was studied in six normal, healthy volunteers to predict its pharmacokinetic profile in man. Maximum absorption of medifoxamine occurred at pH 8, which is close to its pKa (8.5).

Comparative protection efficiency of UVA- and UVB-induced tans against erythema and formation of endonuclease-sensitive sites in DNA by UVB in human skin.

UVA- and UVB-induced tans which were visually identical with each other were induced in separate sites on the lower back of 5 normal human volunteers of good tanning ability. Tanning was achieved by 4 exposures to UVA and UVB administered over an 8-day period. One week after the last exposure the protection afforded by the two types of tan against UVB-induced erythema and against UVB-induced DNA damage was measured. Protection against erythema was measured by comparison of the minimal erythema doses of UVB in tanned and untanned skin. Protection against DNA damage was assessed by comparing the numbers of endonuclease-sensitive sites in epidermal DNA extracted from biopsies taken from tanned and untanned sites exposed to the same dose of UVB. The UVB tans conferred significant protection (mean 2.98-fold) against UVB-induced erythema. UVA tans were not associated with significant protection (mean 1.4-fold). In contrast, both UVA- and UVB-induced tans were associated with a similar reduction in yield of endonuclease-sensitive sites in epidermal DNA (in UVA tan to 47% and in UVB tan to 45% of the yield in untanned skin). Protection conferred by the tans against erythema was therefore not paralleled by protection against DNA damage.

Effect of flecainide on cardiac output.

The effect of intravenous flecainide (2 mg/kg) on cardiac output was evaluated by a dye dilution method in six healthy nonsmokers. The study was of a double-blind, crossover, placebo-controlled, randomized, and balanced design. Cardiac output, heart rate, and stroke volume were measured 0, 10, 30, 60, 90, 120, 150, 180, and 240 minutes after the beginning of the flecainide infusion. Flecainide reduced cardiac output and stroke volume during the first 90 minutes and heart rate increased during the first 30 minutes after flecainide. Visual analog scales for alertness and dry mouth were determined 0, 10, 60, and 240 minutes after dosing. Alertness was reduced 60 minutes after flecainide, but there was no increase in dryness of the mouth.

The effect of a new antiarrhythmic agent, flecainide acetate, on systolic time intervals.

We investigated the effect of flecainide on systolic time intervals in 6 healthy subjects. A randomized, double blind, cross-over study was designed using flecainide (2 mg/kg), propranolol (0.2 mg/kg) or saline. In comparison with placebo, flecainide increased heart rate, diastolic BP, QT-Index, QS2-Index, PEP, and reduced LVET-Index (p less than 0.05). It caused a significant increase in PR interval, QRS duration, and PEP/LVET (p less than 0.01). Propranolol, compared with placebo, decreased heart rate and systolic BP (p less than 0.05). It increased PR interval, PEP, and PEP/LVET ratio (p less than 0.05). In comparison with propranolol, flecainide increased heart rate and QRS duration (p less than 0.01, p less than 0.001 respectively). It increased diastolic BP, PR interval, QS2-Index, PEP, and PEP/LVET ratio, and decreased LVET-Index (p less than 0.05). These changes in STI induced by flecainide may be produced by depression of myocardial contractility. Intraventricular conduction delay may be an additional contributing factor.

Recovery of acetylsalicylic acid and indalpine (LM 5008) after buccal partitioning.

The recoveries were compared of acetylsalicylic acid (ASA) and indalpine (1) after buccal absorption in three trained human volunteers. Recovery of the poorly lipid soluble ASA was less than 1% at pH 5 or 8, while that of the lipid soluble I was 19% and 13% at pH 5 and 8 respectively. This difference in recovery may represent a difference in their storage within the buccal mucosa dependent on their different lipid solubilities.

Tissue vitamin E levels and lipofuscin accumulation with age in the mouse.

To study the deposition of lipofuscin (age pigment) in relation to tissue Vitamin E content a small colony of C3H and LAF mice was set up, one-half being fed a supplement of 0.25% w/w Vitamin E. At intervals between 2.5 and 28 months of age samples of the stock were culled to allow the fluorimetric determination of lipofuscin in heart muscle and an assay of Vitamin E levels in liver tissue. Vitamin E levels in the supplemented stock were found to be 4 X those of the controls although dietary intake was 250 X control. Lipofuscin levels of supplemented stock were lower than controls throughout the lifespan culminating at 28 months when the levels were consistent with the 23 month levels of the controls. Lack of any concomitant effects on longevity, however, raises doubts concerning the relevence of lipid peroxidation to the aging process in the organism as a whole.

Vitamin E--its significance in mouse ageing.

A small colony of C3H/He and LAF1 mice was set up with 50% of all stock being given a dietary supplement of 0.25% w/w vitamin E to study the range of ageing variables over which anti-oxidant administration has an effect. An increase in mean but not maximum lifespan with vitamin E was attributable to fewer fatalities early in life. This may have been due to low anti-oxidant levels in the controls. Lower fatal tumour incidence in both strains and a decrease in collagen content of LAF mice were noted. Lipofuscin levels in heart tissue were, as expected, reduced but the significance of lipid peroxidation to ageing of the organism is questioned.

Changes in skinfold thickness with increasing age.

Skinfold thickness measurements on 626 normal subjects of both sexes between the ages of 5 months and 73 years demonstrated a tri-phasic relationship between loss of skin thickness and age. Measurements for infants of either sex were indistinguishable and high, falling rapidly to levels which only changed slightly over the period 20-60 years. Thereafter, massive loss of skin-thickness occurred in both sexes. Throughout the major portion of adult life the values obtained for females were significantly lower than those for males. It is suggested that the three phases may be associated with progressive dehydration, failure to synthesize collagen and frank degradation of the collagen of the dermis.

The effects of vitamin E on mouse fitness and survival.

A small colony of C3H/He and LAF1 mice, of which 50% were receiving a diet supplemented with vitamin E (0.25%, w/w, dl-alpha-tocopherol), was set up for investigation of the reported action of antioxidants on increasing longevity. It was found that vitamin E exerted no effect on maximum longevity, but increased the numbers of both genotypes surviving to 24 months. The beneficial effects appeared to act by reducing the incidence of fatal tumours late in life and by counteracting a debilitating condition early in life. It is suggested that this debility may have resulted from low antioxidant in the control diet. The relevance of free radicals to ageing is questioned.

Clinical pharmacological studies with LM 5008, a new antidepressant.

A spectrofluorometric assay has been developed for the measurement of LM 5008, a novel antidepressant, in biological fluids. A buccal absorption study in 12 healthy subjects showed increased uptake of LM 5008 under higher pH conditions. Single dose administration of 25 mg orally to 3 volunteers showed peak levels after 2 to 3 hours. Pharmacodynamic studies in 12 volunteers comparing 25 mg LM 5008, 1 mg atropine and placebo showed that pupil diameter increased significantly after LM 5008 compared with atropine and placebo, but no other significant anticholinergic effect was demonstrated. Atropine showed a significant decrease in saliva production and in eccrine sweating and a significant increase in dryness of the mouth and in pupil diameter compared with placebo. The distance of the near point tended to increase during atropine treatment but only significantly at 6 hours. LM 5008 produced no significant change in these measurements.