Observational cohort study of the effect of a single lubricant exposure during transvaginal ultrasound on cell-shedding from the vaginal epithelium.

The outer layers of the vaginal epithelium (VE) are important because they accumulate glycogen which, under optimal conditions, Lactobacillus spp. consume to grow and acidify the vaginal microenvironment with lactic acid. We hypothesized that exposure to lubricant, for example in the conduct of a transvaginal ultrasound (TVUS), may contribute to the shedding of mature epithelial cells, exposing immature cells. Cervicovaginal fluid (CVF) was sampled at four time points by menstrual cup (Softdisc™) from 50 women referred for TVUS, during which a controlled volume of lubricant was applied to the TVUS wand. Samples were collected (1) immediately before TVUS and (2) 6-12 hours, (3) within one week, and (4) two weeks after TVUS. Clinical vaginal lubricants are similar to commercial lubricants, and often have a high osmolality or pH, and contain bactericides such as methylparaben and propylparaben. The number and maturity of epithelial cells in each CVF sample were measured by quantitative and differential fluorimetry (maturity index, MI). Comparisons of cell-counts and maturity were made by paired Wilcoxon signed-rank tests. Among women with a high pre-TVUS MI (> 3), there was a decrease in median cell-count and mean MI in the sample collected 6-12 hours after TVUS (p<0.001, n = 26 and p < 0.001, n = 26, respectively). For these women, cell-count and MI remained lower in the sample collected within the subsequent week (p<0.001, n = 29 and p<0.01, n = 29, respectively), and MI remained lower in the sample collected within two weeks of TVUS (p<0.01, n = 25), compared to the pre-TVUS sample. Among participants with a low pre-TVUS MI (< 3), cell-count was higher in the sample collected within two weeks of TVUS compared to the pre-TVUS sample (p = 0.03, n = 15), but no significant changes in MI were observed. Results were similar when restricted to reproductive-age women. This preliminary data indicates hypertonic vaginal lubricants may increase vaginal epithelial cell shedding.

Asymptomatic Bacterial Vaginosis Is Associated With Depletion of Mature Superficial Cells Shed From the Vaginal Epithelium.

Previous studies have described bacterial vaginosis (BV) as associated with increased cell-shedding from the cervicovaginal epithelium. Cell-shedding in excess of cell-proliferation is thought to decrease epithelial barrier function and increase susceptibility to infection. This study evaluated the number of shed cells in mid-vaginal smears from women with a diagnosis of symptomatic BV (sBV, n = 17), asymptomatic BV (aBV, n = 71), or no BV (n = 104) by Amsel criteria. The sBV smears contained significantly more shed cells (median 158/100X field) than no BV smears (median 91/100X field), p = 7.2e-9. However, we observed that aBV smears contained significantly fewer shed cells (median 35/100X field) than no BV smears, p = 22.0e-16. The sizes of cell-aggregates (cells shed in sometimes multilayered sections with intact cell-cell attachments) followed the same pattern. Cell-aggregates in sBV smears were significantly larger (median ~220,000 µm2) than those in no BV smears (median ~50,000 µm2), p = 1.8e-6, but cell-aggregates in aBV smears were significantly smaller (median ~7,000 µm2) than those in no BV smears, p = 0.0028. We also compared the superficial cell index (SCI), a measure of cervicovaginal epithelial cell maturity, in no BV and aBV smears with relatively low numbers of shed cells (≤50/100X field). The SCI of no BV smears was significantly higher (median 0.86) than that of aBV smears (median 0.35), p = 4.3e-98, suggesting a depletion of mature cells with exposure and shedding of underlying immature cells in aBV with low number of shed cells. These results indicate that aBV may contribute disproportionately to the increased susceptibility to reproductive tract infections associated with BV. Our findings remained true when considering only those smears in which the microbiota comprised a diverse set of strict and facultative anaerobic bacteria [Community State Type IV (n = 162)], thus excluding those dominated by Lactobacillus spp. This is consistent with our developing hypothesis that high-shedding sBV and low-shedding aBV could be temporally separated phases of the same condition, rather than two separate forms of BV. These findings might inform future work on clinical management of symptomatic and asymptomatic bacterial vaginosis.

Rapid transport of large polymeric nanoparticles in fresh undiluted human mucus.

Nanoparticles larger than the reported mesh-pore size range (10-200 nm) in mucus have been thought to be much too large to undergo rapid diffusional transport through mucus barriers. However, large nanoparticles are preferred for higher drug encapsulation efficiency and the ability to provide sustained delivery of a wider array of drugs. We used high-speed multiple-particle tracking to quantify transport rates of individual polymeric particles of various sizes and surface chemistries in samples of fresh human cervicovaginal mucus. Both the mucin concentration and viscoelastic properties of these cervicovaginal samples are similar to those in many other human mucus secretions. Unexpectedly, we found that large nanoparticles, 500 and 200 nm in diameter, if coated with polyethylene glycol, diffused through mucus with an effective diffusion coefficient (D(eff)) only 4- and 6-fold lower than that for the same particles in water (at time scale tau = 1 s). In contrast, for smaller but otherwise identical 100-nm coated particles, D(eff) was 200-fold lower in mucus than in water. For uncoated particles 100-500 nm in diameter, D(eff) was 2,400- to 40,000-fold lower in mucus than in water. Much larger fractions of the 100-nm particles were immobilized or otherwise hindered by mucus than the large 200- to 500-nm particles. Thus, in contrast to the prevailing belief, these results demonstrate that large nanoparticles, if properly coated, can rapidly penetrate physiological human mucus, and they offer the prospect that large nanoparticles can be used for mucosal drug delivery.

S100A6 is a negative regulator of the induction of cardiac genes by trophic stimuli in cultured rat myocytes.

S100A6 (calcyclin), a member of the S100 family of EF-hand Ca2+ binding proteins, has been implicated in the regulation of cell growth and proliferation. We have previously shown that S100B, another member of the S100 family, is induced postinfarction and limits the hypertrophic response of surviving cardiac myocytes. We presently report that S100A6 expression is also increased in the periinfarct zone of rat heart postinfarction and in cultured neonatal rat myocytes by treatment with several trophic agents, including platelet-derived growth factor (PDGF), the alpha1-adrenergic agonist phenylephrine (PE), and angiotensin II (AII). Cotransfection of S100A6 in cultured neonatal rat cardiac myocytes inhibits induction of the cardiac fetal gene promoters skeletal alpha-actin (skACT) and beta-myosin heavy chain (beta-MHC) by PDGF, PE, AII, and the prostaglandin F2alpha (PGF2alpha), induction of the S100B promoter by PE, and induction of the alpha-MHC promoter by triiodothyronine (T3). By contrast, S100B cotransfection selectively inhibited only PE induction of skACT and beta-MHC promoters. Fluorescence microscopy demonstrated overlapping intracellular distribution of S100B and S100A6 in transfected myocytes and in postinfarct myocardium but heterodimerization of the two proteins could not be detected by co-immunoprecipitation. We conclude that S100A6 may function as a global negative modulator of differentiated cardiac gene expression comparable to its putative role in cell cycle progression of dividing cells.

Malignant phaeochromocytoma metastasising to the breast.

BACKGROUND: Nearly one-quarter of metastatic tumours in the breast are from an occult extramammary tumour, usually a lung carcinoma. AIM: To report on a patient with a history of metastatic malignant phaeochromocytoma and a breast mass. RESULT: A 54-year-old female presented with a right breast mass. At the age of 32, she had presented with a phaeochromocytoma. The staining of the breast mass was comparable with that of her original adrenal tumour. CONCLUSION: This is the first published case of a phaeochromocytoma metastasising to the breast, and demonstrates the challenge that extramammary tumours in the breast can pose for the pathologist.

Soluble adhesion molecules (E-selectin, ICAM-1 and VCAM-1) in breast carcinoma.

Adhesion molecules are important in cell-cell and cell-basement membrane interactions. They are intimately involved in inflammatory reactions and a role in tumour progression has been postulated. E-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) play a role in cell adhesion to the vascular endothelium, and may have a role in tumour cell dissemination. Soluble forms of these molecules have been described and this study was established to examine these adhesion molecules in patients with breast carcinoma. Serum was obtained from 92 patients with breast carcinoma and 31 age-matched patients with benign breast disease. All samples were obtained prior to surgery. Soluble levels of E-selectin, ICAM-1, and VCAM-1 were significantly elevated in patients with Stage 4 disease compared with controls. (E-selectin 88.6 (47.9) versus 51.4 (18.4) ng/ml; P<0.001: ICAM-1 447 (249) versus 244 (79) ng/ml; P<0.001: VCAM-1 779 (159) versus 552 (135) ng/ml; P<0.001 results expressed on mean (SEM) SD placed above this.). The prognostic value of the adhesion molecules was examined. In patients with Stage 2 disease, elevated VCAM-1 was predictive of decreased survival, even when corrected for T and N status. Adhesion molecules are elevated in patients with advanced disease and elevation in VCAM-1 has prognostic significance in patients with breast carcinoma.

An immunohistochemical study of p21 and p53 expression in primary node-positive breast carcinoma.

AIMS: p21, an inhibitor of cyclin-dependent kinase, is involved in the p53 pathway of growth control. Its expression has been linked to cellular differentiation. It has been implicated in p53-mediated growth arrest following DNA damage and in terminally differentiated cells. This study analysed p21 and p53 expression, in a series of node-positive patients with breast carcinoma and examined histopathological parameters of the tumour and the prognostic implications of p21 and p53 expression. METHODS: One hundred and five consecutive patients with node-positive disease and at least 3 years follow-up were identified. Sections were stained for p53 and p21 using monoclonal antibodies. Results were expressed as percentage positive cells, and over 20% considered positive for p53 and over 10% considered for p21. RESULTS: p21 was overexpressed (>10% of cells positive) in 65% of patients and p53 was overexpressed (>20% of cells positive in 68%. The mean (SEM) level of p21 staining was 5.7(0.8)% and was 54.9(4.0)% for p53. There was no correlation between p21 and p53 expression (r=0.071 P=0.5). There were no significant differences in demographic criteria between patients that were p21 positive or negative and p53 positive or negative. There were no significant differences in tumour type, grade or stage between the groups. p21 expression did not have prognostic significance; however, p53 positivity was associated with a worse prognosis, which remained when controlled for stage. CONCLUSIONS: This study demonstrated p21 overexpression in 65% of patients with node-positive breast carcinoma. Levels did not correlate with p53 status and unlike p53 failed to have prognostic significance.

Advantages of a posterior fourchette incision in anal sphincter repair.

PURPOSE: Delayed repair of obstetric-related anal sphincter injury remains problematic, and perineal wound breakdown is common. The aim of this study was to assess the outcome after overlap anal sphincter repair and to determine the advantages, if any, of a posterior fourchette incision (n = 18) compared with a conventional perineal incision (n = 32). METHODS: Fifty females of mean parity 2.8 (standard deviation, 1.6) underwent repair in a five-year period. The mean follow-up was 23 months. Assessment was by anal vector manometry, endoanal ultrasound, and continence scoring. RESULTS: Functional outcomes were similar in the two groups. Repair increased squeeze-pressure increment and improved continence scores in both groups. Postoperative wound complications were fewer when a posterior fourchette incision was used compared with a perineal incision (11 vs. 44 percent, respectively; P < 0.05). CONCLUSIONS: Delayed anal sphincter repair improves continence. A posterior fourchette approach is associated with fewer postoperative wound complications without compromising the quality of repair and the functional outcome.

The prognostic significance of CD44s and CD44v6 expression in stage two breast carcinoma: an immunohistochemical study.

AIMS: Expression of the v6 variant isoform of CD44 has been causally associated with the development of metastases. This study, using immunohistochemical techniques, examined the prognostic significance of CD44s and CD44v6 expression. METHODS: A cohort of 109 women presenting with stage 2 breast cancer, with a minimum follow-up of 5 years, were assessed. RESULTS: Eighty percent of patients demonstrated CD44v6 expression on immunohistochemical studies. CD44v6 expression in tissue sections was found to be independent of age, tumour size, grade, and lymph-node status. No significant association was demonstrated between CD44v6 expression and either disease-free or overall survival. Similar findings were observed for CD44s. CONCLUSIONS: CD44s and CD44v6 do not appear to be useful as prognostic indicators in early breast cancer. The increased expression of variant CD44 isoforms seen in breast neoplasia may merely be a marker for loss of control of alternative splicing within tumour tissue.

Prognostic implications of p53 and bcl-2 expression in 108 women with stage two breast cancer.

BACKGROUND: The mortality and morbidity of patients with breast cancer can vary even between individuals with similar histological stage at diagnosis. Identification of those individuals with prognostically poorer tumours is an essential prerequisite in planning adjuvant therapies. Some prognostic indices of tumour size, grade, oestrogen receptor status and nodal status are well established. AIM: The aim of this study was to examine the prognostic role of information relating to proto-oncogene and tumour suppressor gene expression. METHODS: 108 women with stage II breast cancer were studied. Tumour expression of p53 and bcl-2 were scored and then correlated with recurrence and mortality. RESULTS: We have shown that individuals poorly expressing bcl-2 in their tumours have a poorer disease-free and overall survival than those who express bcl-2. When p53 was strongly expressed, it was associated with poorer disease-free and overall survival. CONCLUSION: The profiling of individual tumour genetic expression of proto-oncogenes may allow for more specific identification of patients at higher risk of recurrence in breast cancer.

A randomized, controlled trial of methotrexate versus placebo in early diffuse scleroderma.

OBJECTIVE: Early diffuse scleroderma (systemic sclerosis; SSc) has no proven treatment. This study was undertaken to examine the efficacy of methotrexate (MTX) in improving the skin and other disease parameters in early diffuse SSc. METHODS: Seventy-one patients with diffuse SSc of <3 years' duration were enrolled in a multicenter, randomized, placebo-controlled, double-blind trial. Thirty-five patients were treated with MTX and 36 with placebo. Treatment was administered for 12 months. The primary outcome measures were skin score (as determined with 2 different indices) and physician global assessment. RESULTS: At baseline, there were no statistically significant differences in skin scores, carbon monoxide diffusing capacity (DLco), physician global assessment, or other secondary outcome measurements between the 2 treatment groups. At study completion, results slightly favored the MTX group (mean +/- SEM modified Rodnan skin score 21.4+/-2.8 in the MTX group versus 26.3+/-2.1 in the placebo group [P < 0.17]; UCLA skin score 8.8+/-1.2 in the MTX group versus 11.0+/-0.9 in the placebo group [P < 0.15]; DLco in the MTX group 75.7+/-4.6 versus 61.8+/-3.4 in the placebo group [P < 0.2]). In addition, physician global assessment results favored MTX (P < 0.035), whereas patient global assessment did not differ significantly between groups. When between-group differences for changes in scores from baseline to 12 months were examined using intent-to-treat methodology, MTX appeared to have a favorable effect on skin scores (modified Rodnan score -4.3 in the MTX group versus 1.8 in the placebo group [P < 0.009]; UCLA score -1.2 in the MTX group versus 1.2 in the placebo group [P < 0.02]), but differences in the degree of change in the DLco and physician global assessment were not significant. For the UCLA skin score, these differences in results were not statistically significant after adjustment for baseline differences in sex distribution and steroid use. Dropout rates were similar in the 2 groups. CONCLUSION: Although results of this trial demonstrated a trend in favor of MTX versus placebo in the treatment of early diffuse SSc, the between-group differences were small and the power to rule out false-negative results was only 50%. Our findings do not provide evidence that MTX is significantly effective in the treatment of early diffuse SSc.

Timing of pre-emptive tenoxicam is important for postoperative analgesia.

PURPOSE: In this prospective randomized study, a comparison was made between the efficacy of 20 mg tenoxicam, administered either, 30 min preoperatively or at induction of anesthesia, for the relief of postoperative pain in patients undergoing ambulatory breast biopsy. METHODS: Seventy-three patients were recruited and all received a standard anesthetic consisting of induction with 2 mg x kg(-1) propofol followed by 5 microg x kg(-1) alfentanyl. No premedication was administered and at the end of the procedure the wounds were infiltrated with 10 ml of bupivacaine (0.5%). Patients were randomized to receive 20 mg tenoxicam intraveneously either 30 min before surgery or at induction of anesthesia. RESULTS: Demographic criteria were similar in both groups. There were differences in pain scores at 30, 60, 120 and 240 min postoperatively (VAS at 30 min 3.2 +/- 1.2 vs 5.5 +/- 1.8; P < 0.001: VAS at 60 min 1.8 +/- 1.2 vs 3.7 +/- 1.9; P < 0.001: VAS at 120 min 0.9 +/- 0.9 vs 1.7 +/- 1.0; P = 0.003: VAS at 240 min 0.5 +/- 0.5 vs 1.1 +/- 0.8; P < 0.001: Expressed as mean +/- SD). There was a difference in the number of patients requiring additional analgesia, in the first four hours postoperatively (12 (33%) vs 27 (73%); P = 0.001) and a difference in the time to additional analgesia in these patients (87.5 +/- 32.5 vs 55.0 +/- 26.8 min; P = 0.002). CONCLUSION: Early administration of pre-emptive tenoxicam 30 min before induction of anesthesia improves postoperative analgesia in patients undergoing ambulatory breast biopsy.

Preemptive bupivacaine offers no advantages to postoperative wound infiltration in analgesia for outpatient breast biopsy.

BACKGROUND: Adequate analgesia is important after surgery and in particular after ambulatory surgery. Preemptive administration of analgesics, ie, prior to commencing surgery, has many theoretical advantages. METHODS: In this prospective randomized study, the use of preincisional bupivacaine was compared with a postincision dose for the relief of postoperative pain, in 74 patients undergoing day-case breast biopsy. RESULTS: Demographic criteria were similar in both groups. There were no differences in pain scores postoperatively on the visual analog scale (VAS): VAS at 30 minutes 4.5 ([SD] 2.4) versus 4.7 (1.9); P = not significant (NS); VAS at 60 minutes 3.3 (2. 3) versus 3.6 (2.2); P = NS; VAS at 120 minutes 1.9 (1.7) versus 2.5 (2.0); P = NS; VAS at 240 minutes 0.9 (1.0) versus 1.3 (1.4); P = NS. There was no difference in the number of patients requiring additional analgesia: 13 (36%) versus 18 (47%); P = NS. Nor was there a difference in the time to additional analgesia: 55.0 (37.8) versus 55.3 (39.2) minutes; P = NS. CONCLUSIONS: The administration of local anaesthesia prior to starting surgery does not appear to have any advantage over its postoperative administration in patients undergoing ambulatory breast biopsy.

An assessment of the value of intraperitoneal meperidine for analgesia postlaparoscopic tubal ligation.

UNLABELLED: Patients undergoing laparoscopic procedures may experience postoperative pain. The intraperitoneal (IP) administration of drugs is controversial but has proven effective in some studies for the relief of postoperative pain. However, some investigators have not been able to confirm the analgesic efficacy of IP local anesthetics. The administration of IP opioids for the relief of postoperative pain has received little attention. At the end of laparoscopic tubal ligation, 100 patients received 80 mL of 0.125% bupivacaine with 1:200,000 epinephrine IP and 50 mg of meperidine either IP or IM. Postoperative pain scores were measured at rest and with movement. Pain scores were significantly lower in the group receiving the IP meperidine both at rest (P: < 0.01) and with movement (P: < 0.05). We conclude that the combination of intraperitoneal bupivacaine and intraperitoneal meperidine was better than the combination of IP bupivacaine and IM meperidine for postoperative analgesia in patients undergoing laparoscopic tubal ligation. IMPLICATIONS: The combination of bupivacaine and meperidine delivered to the intraperitoneal cavity proved superior to equivalent doses of intraperitoneal bupivacaine and IM meperidine for postoperative pain relief in patients undergoing laparoscopic tubal ligation. Intraperitoneal delivery of analgesia proved effective in this study and merits further study and more widespread use.

Enhanced calcium transients in glial cells in neonatal cerebellar cultures derived from S100B null mice.

S100B is the major low-affinity Ca(2+)-binding protein in astrocytes. In order to study the role of S100B in the maintenance of Ca(2+) homeostasis, we generated S100B null mice by a targeted inactivation of the S100B gene. Absence of S100B expression was demonstrated by Northern and Western blotting for S100B mRNA and protein, respectively, and immunoperoxidase staining of sections of various brain regions. S100B null mice were viable, fertile, and exhibited no overt behavioral abnormalities up to 12 months of age. On the basis of light microscopy and immunohistochemical staining, there were no discernable alterations in the distribution and morphology of astrocytes or neurons in sections of adult brains of these mice. Astrocytes in cerebellar cultures derived from 6-day-old S100B null mice exhibited enhanced Ca(2+) transients in response to treatment with KCl or caffeine. On the other hand, granule neurons, in the same cultures, exhibited normal Ca(2+) transients in response to treatment with KCl, caffeine, or N-methyl-d-aspartate. These results demonstrate a specific decrease in Ca(2+)-handling capacity in astrocytes derived from S100B null mice and suggest that S100B plays a role in the maintenance of Ca(2+) homeostasis in astrocytes.