Adsorption mechanisms of L-Glutathione on Au and controlled nano-patterning through Dip Pen Nanolithography.

Dip Pen Nanolithography technique has been employed for patterning L-Glutathione tripeptide (l-y-glutamyl-l-cysteinyl-glycine) nanostructures at specific locations on metallic Au(111) substrate. The formed supramolecular architectures were designed through straight lines and dots serving as precursors for building blocks assemblies in nano-bio-electronics applications or as template structures for functionalized particles in the form of host-guest networks. Tween 20 polyoxyethylene surfactant concentrations ranging from 0.005 to 0.1% (v/v) into initial l-Glutathione tripeptide (2 mg mL(-1)) ink solutions were sequentially tested for the improvement of the ink delivery process and to assure an optimum uniformity and homogeneity over the patterned space. A strong relationship was found between the coated atomic force microscope (AFM) cantilever within the highly effective Tween 20 activator adjuvant and the molecular diffusion along concentration gradients. An increase in the driving force for ink transport from the AFM tip has been demonstrated within the highest 0.1% (v/v) TW 20 surfactant concentration, favoring the patterning of GSH molecules routinely with sub-100 nm resolution. Self-assembled monolayers of GSH were also fabricated and characterized in the light of X-ray photoemission spectroscopy (XPS) and ellipsometric optical measurements. Adsorption from water of l-Glutathione to the gold substrate is proven to be made by the thiol group of cysteine. Theoretical DFT approaches were applied for quantum chemical studies dedicated to electronic processes underneath molecular GSH/Au(111) systems.

The miR-17∼92 family regulates the response to Toll-like receptor 9 triggering of CLL cells with unmutated IGHV genes.

Chronic lymphocytic leukemia (CLL) cells from clinically aggressive cases have a greater capacity to respond to external microenvironmental stimuli, including those transduced through Toll-like-receptor-9 (TLR9). Concomitant microRNA and gene expression profiling in purified CLL cells (n=17) expressing either unmutated (UM) or mutated (M) IGHV genes selected microRNAs from the miR-17∼92 family as significantly upregulated and in part responsible for modifications in the gene expression profile of UM CLL cells stimulated with the TLR9 agonist CpG. Notably, the stable and sustained upregulation of miR-17∼92 microRNAs by CpG was preceded by a transient induction of the proto-oncogene MYC. The enforced expression of miR-17, a major member from this family, reduced the expression of the tumor suppressor genes E2F5, TP53INP1, TRIM8 and ZBTB4, and protected cells from serum-free-induced apoptosis (P ≤ 0.05). Consistently, transfection with miR-17∼92 family antagomiRs reduced Bromo-deoxy-uridine incorporation in CpG-stimulated UM CLL cells. Finally, miR-17 expression levels, evaluated in 83 CLL samples, were significantly higher in UM (P=0.03) and ZAP-70(high) (P=0.02) cases. Altogether, these data reveal a role for microRNAs of the miR-17∼92 family in regulating pro-survival and growth-promoting responses of CLL cells to TLR9 triggering. Overall, targeting of this pathway may represent a novel therapeutic option for management of aggressive CLL.

Exposure of B cell chronic lymphocytic leukemia (B-CLL) cells to nutlin-3 induces a characteristic gene expression profile, which correlates with nutlin-3-mediated cytotoxicity.

By analyzing the cDNA obtained from 16 B-cell chronic lymphocytic leukemia (B-CLL) patient samples, we found that Nutlin-3, a small molecule inhibitor of MDM2/p53 interaction, induced a characteristic gene expression profile (GEP) signature in 13 out of 16 B-CLL samples. The lack of Nutlin-3-induced GEP signature in 3 out of 16 B-CLL samples was not due to p53 deletion and/or mutation, as demonstrated by FISH analysis and p53 sequencing. Of note, the 3 B-CLL samples in which Nutlin-3 did not elicit the GEP signature were also less susceptible to Nutlin-3-mediated cytotoxicity with respect to the remaining 13 B-CLL samples. However, the partial lack of response in these p53 wild-type B-CLL samples was not due to defects in the ability of Nutlin-3 to promote p53 induction, as confirmed by the rapid accumulation of p53 protein at Western blot analysis in response to Nutlin-3 in all samples examined. Upon exposure to Nutlin-3, the genes up-regulated with the highest score in the majority of B-CLL cells were all known p53-target genes, including genes involved in apoptotic pathways, such as FAS and BAX, as well as MDM2. Taken together, our data indicate that the ability of Nutlin-3 to induce a characteristic GEP signature correlates with its cytotoxic potential in p53 wild-type B-CLL cells. However, in some p53 wild-type B-CLL samples, the response to Nutlin-3 cannot be predicted on the basis of FISH analysis or p53 sequencing.

Comparison of two fertility-sparing approaches for bilateral borderline ovarian tumours: a randomized controlled study.

BACKGROUND: During the childbearing years, the standard fertility-sparing treatment for bilateral borderline ovarian tumours (BOTs) is the unilateral oophorectomy plus controlateral cystectomy. The aim of the present study was to compare the effects of two laparoscopic fertility-sparing surgical procedures for the treatment of bilateral BOTs on recurrence and fertility in young women who desire to conceive as soon as possible. METHODS: Thirty-two women affected by bilateral early-stage BOTs who desired to conceive were randomized to receive bilateral cystectomy (experimental group, n=15) or oophorectomy plus controlateral cystectomy (control group, n=17). At the first recurrence after childbearing completion, each patient was treated with non-conservative standard treatment. Recurrences and reproductive events were recorded. RESULTS: After a follow-up period of 81 months (19 inter-quartile; 60-96 range), the cumulative pregnancy rate (CPR) (14/15 versus 9/17; P=0.003) and the cumulative probability of first pregnancy (P= 0.011) were significantly higher in the experimental than in control group. No significant (P=0.358) difference between groups was detected in cumulative probability of first recurrence. CONCLUSIONS: The laparoscopic bilateral cystectomy followed by non-conservative treatment performed at the first recurrence after the childbearing completion is an effective surgical strategy for patients with bilateral early-stage BOTs who desire to conceive as soon as possible.

Ultrasound-assisted laparoscopic cryomyolysis: two- and three-dimensional findings before, during and after treatment.

OBJECTIVE: To investigate the role of two- and three-dimensional (2D and 3D) ultrasound and power Doppler before, during and after surgery in monitoring the effects of uterine fibroid laparoscopic cryomyolysis. METHODS: This prospective study involved 10 premenopausal patients with a sonographic diagnosis of a single subserosal and/or intramural uterine myoma, who underwent laparoscopic cryomyolysis. All patients suffered from symptoms due to fibroids (menometrorrhagia, bulk-related symptoms, pelvic pain). During laparoscopy transvaginal sonography was performed to guide the insertion of the cryoprobe, monitor extension of the ice-ball and evaluate the reduction of the blood supply of the myoma. All patients underwent 2D and 3D sonographic and power Doppler imaging evaluation of the myoma 1 week before treatment, during cryomyolysis, and 1, 3 and 6 months after treatment; size, echostructure and vascularization were recorded. RESULTS: With cryomyolysis, we achieved selective vessel and tissue damage within the fibroid alone. Eight patients were free of symptoms and two had improved after 3 months. Progressive shrinkage of the treated myoma was observed during follow-up with a reduction percentage after 1 month of 22.2%, after 3 months of 37.5% and after 6 months of 52.6%. After cryomyolysis a significant reduction in central blood flow of the myoma was observed. 2D and 3D power Doppler evaluation of vascularization did not differ although subjectively findings were best evaluated by 3D images. CONCLUSION: Sonography can aid the safe accomplishment of cryomyolysis by assessing myomata preoperatively, guiding the freezing procedure during laparoscopy, and helping to monitor postoperative progress. The use of ultrasound in this new treatment of fibroids will permit the physician to modulate and individualize treatment.

Hysteroscopic findings in 344 women with recurrent spontaneous abortion.

STUDY OBJECTIVE: To evaluate the prevalence of different anatomic factors in women with recurrent spontaneous abortion (RSA). DESIGN: Retrospective analysis over 9 years (Canadian Task Force classification II-2). SETTING: University hospital-affiliated endoscopic unit. PATIENTS: Three hundred forty-four consecutive patients with RSA and 922 controls referred for abnormal uterine bleeding. INTERVENTION: Diagnostic hysteroscopy. MEASUREMENTS AND MAIN RESULTS: Major and minor uterine mullerian abnormalities (septate, unicornuate uteri) were found significantly more often in women with RSA than in controls (32% vs 6%, p <0.001). The frequency of acquired uterine anomalies (submucous myomas, polyps) was significantly higher in controls (32% vs 9%, p <0.001). No significant differences were observed between groups in frequency of adhesions (4% vs 2%). CONCLUSION: Major mullerian uterine abnormalities are associated with RSA, and minor uterine anomalies may be correlated with an increased risk of recurrent miscarriage.

TNFalpha expression in hyperplastic endometrium.

PROBLEM: Tumor necrosis factor-alpha (TNFalpha) is produced by the endometrium, and it has been shown that this cytokine has cyclic variations through the menstrual cycle. METHOD OF STUDY: In this study we assessed TNFalpha, estradiol and progesterone levels in the endometrium and serum of 21 patients with recent abnormal uterine bleeding (AUB). Eleven women showed histological diagnosis of endometrial hyperplasia, and ten women showed normal endometrium. RESULTS: The TNFalpha value in the serum of patients with hyperplasia and in normal endometrium did not show significant differences (64.8 +/- 21.4 vs 77.6 +/- 14.8 pg). The amounts of TNFalpha released by hyperplastic endometrial tissue were significantly higher than in control patients (258.8 +/- 78.0 vs 140.6 +/- 79.8 pg/g; P < 0.001). Immunohistochemistry of hyperplastic endometria showed a stronger staining than normal ones. Serum E2 levels in patients with hyperplastic endometrium were 49.5 +/- 31.6 ng/mL, and in controls were 107.3 +/- 66.0 ng/mL. The levels of estradiol released by hyperplastic endometria were significantly lower than in normal endometria (152.7 +/- 60.0 vs 279.9 +/- 178.7 ng/g; P < 0.003). CONCLUSIONS: Hyperplastic endometria produce higher amounts of TNFalpha with respect to controls. These findings may explain the frequent bleeding observed in the patients with endometrial hyperplasia, since TNFalpha promotes apoptosis and endometrial shedding.

Is local anesthesia an affordable alternative to general anesthesia for minilaparoscopy?

STUDY OBJECTIVE: To determine if minilaparoscopy under local anesthesia is at least as reliable and affordable as that performed under general anesthesia. DESIGN: Prospective, randomized study (Canadian Task Force classification I). SETTING: University-affiliated hospital. PATIENTS: One hundred sixty-four consecutive women evaluated for infertility. INTERVENTION: Diagnostic minilaparoscopy performed after women were randomized to receive general or local anesthesia with conscious sedation. MEASUREMENTS AND MAIN RESULTS: Levels of postoperative pain measured by visual analog scale; volume of CO(2) used; length of procedure, complete pelvic evaluation, and hospitalization; complications; and pathologic diagnosis were evaluated. The groups were comparable in age, years of infertility, and symptoms. For women receiving local anesthesia, 5.5% required general anesthesia to complete the procedure. Women in both groups required postoperative analgesics. The groups had no statistically significant differences in pain level 1 hour after the procedure, number of complications, and pelvic pathology. Patients who had local anesthesia required a smaller volume of CO(2) (p <0.01) and their hospitalization was significantly shorter (p <0.01). However, in 15% of these women pelvic visualization was incomplete, compared with 7.2% in the general anesthesia group. CONCLUSION: Minilaparoscopy performed under local anesthesia was as reliable and affordable as when performed under general anesthesia.

Pain mapping during minilaparoscopy in infertile patients without pathology.

STUDY OBJECTIVE: To draw a map of pelvic pain and quantify the level of provoked pain during minilaparoscopy under local anesthesia and conscious sedation. DESIGN: Observational study (Canadian Task Force classification II-2). SETTING: University hospital. PATIENTS: Twenty infertile women. INTERVENTIONS: Minilaparoscopy was performed under local anesthesia and conscious sedation, and cognitive performance was evaluated with the Rey auditory verbal learning task. MEASUREMENTS AND MAIN RESULTS: The diagnostic procedure was performed with one 2-mm micrograsper and one 2-mm microprobe to evaluate the pelvis. In particular we grasped utero-ovarian ligaments; we touched, grasped, and distended fallopian tubes with blue dye; we moved the uterus with a manipulator inserted at the cervix; and we touched and grasped bowel and omentum. Level of pain was recorded on a visual analog scale. Patients had no pathologic findings, including minimal endometriosis and pelvic adhesions. The highest level of pain was recorded when we distended the tubes. No pain was elicited when we touched and grasped ovary, omentum, and bowel. In 10% of women when we stretched the tubo-ovarian ligament we provoked a minimal vagal reaction. CONCLUSION: Minilaparoscopy under conscious sedation for pelvic pain mapping in women without pain or pathology revealed consistently negative findings, validating the value of this measurement. (J Am Assoc Gynecol Laparosc 6(1):51-54, 1999)

Ultrasonographic prediction of the efficacy of GnRH agonist therapy before laparoscopic myomectomy.

STUDY OBJECTIVE: To assess ultrasonographic prediction of the efficacy of administration of a gonadotropin-releasing hormone (GnRH) analog before laparoscopic myomectomy. DESIGN: Prospective, randomized study of women treated consecutively from September 1994 to July 1996 (Canadian Task Force classification I). SETTING: Endogyn Service, Private Endoscopic Associates, Naples, and Department of Gynecologic and Pediatric Sciences, Reggio Calabria University, Catanzaro, Italy. PATIENTS: Sixty-seven infertile women with symptomatic uterine myomata, mainly intramural, undergoing laparoscopic myomectomy. INTERVENTIONS: Patients were prospectively randomized in two groups. Group A received preoperative administration of two injections of a depot formulation of leuprolide acetate 28 days apart, and group B underwent direct surgery. In each group we studied the number, diameter, and echogenicity of larger fibroids; resistance index of uterine arteries and myoma vessels; operating time; and blood loss. MEASUREMENTS AND MAIN RESULTS: The two groups did not significantly differ in baseline ultrasonographic parameters. Both blood loss (p <0.01) and operating time (p <0.05) were significantly lower in group A. However, operating time was significantly longer when the main myoma was markedly hypoechoic. CONCLUSION: Our data confirm the therapeutic efficacy of administration of a GnRH analog before laparoscopic myomectomy in reducing blood loss and decreasing operating time in all cases except those with markedly hypoechoic fibroids.

Outpatient diagnostic hysteroscopy.

STUDY OBJECTIVE: To evaluate the feasibility, validity, indications, and results of a large series of diagnostic hysteroscopies performed without anesthesia. DESIGN: Retrospective analysis of hysteroscopy charts performed between 1989 and 1996 (Canadian Task Force classification II-2). SETTING: University-affiliated endoscopy unit. PATIENTS: Four thousand consecutive women referred for different indications. INTERVENTIONS: Diagnostic hysteroscopy was performed in 91% of patients without premedication or anesthetics. In some women premedication or general or local anesthesia was required to access the uterine cavity. MEASUREMENTS AND MAIN RESULTS: The success rate, validity indication, complication rate, and number of biopsies were critically evaluated and assessed in relation to increased experience of operators. In 91% of women we accessed the uterine cavity at the first attempt without premedication, whereas 207 (5. 1%) patients required local anesthesia and 99 (2.4%) premedication. Only 1.6% required general anesthesia. In 52% intrauterine pathology was diagnosed and in 21% further surgical treatment was suggested. CONCLUSION: Hysteroscopy was feasible when performed in an outpatient setting without general or local anesthesia in more than 90% of women. The operator's experience seems a key factor both for accurate endometrial evaluation and to reduce failure and endometrial biopsy rates. The low frequency of further surgical treatment justifies performing the procedure in the office.

Pain control after microlaparoscopy.

STUDY OBJECTIVE: To evaluate the efficacy of intraperitoneal subdiaphragmatic instillation of 0.5% lidocaine and 0.5% bupivacaine infiltration of cannula sites to control pain after diagnostic microlaparoscopy. DESIGN: Prospective, randomized study. (Canadian Task Force classification I). SETTING: Day surgery unit of Endogyn Service, Private Endoscopic Associates, Naples, and Department of Gynecologic and Pediatric Sciences, Reggio Calabria University, Catanzaro, Italy. PATIENTS: Forty women treated for infertility. INTERVENTIONS: The treated group received 0.5% intraperitoneal subdiaphragmatic lidocaine 40 ml and 0.5% bupivacaine 5 ml infiltration of cannula insertion sites. The control group received no treatment. In all patients the procedure was performed with atropine 0.5 mg, fentanyl 0.1 mg, droperidol 5 mg, and local anesthesia. Postoperatively, depending on the need, ketoprofene 100 mg or ketorolac 30 mg was administered intramuscularly. MEASUREMENTS AND MAIN RESULTS: Postoperative pain score was evaluated by visual analog scale immediately postoperatively and 1, 3, 6, 12, 24, 36, and 48 hours afterward. The treated group had significantly lower pain scores at the end of surgery and at 1-, 3-, (p <0.01), and 6-hour intervals (p <0.05). No significant differences in scores between groups were observed starting from 6 hours postoperatively. CONCLUSION: Postoperative intraperitoneal lidocaine and bupivacaine infiltration of cannula sites offered a detectable benefit to women undergoing diagnostic microlaparoscopy. The effect was temporary, but induced a significant decrease in the postoperative pain for approximately 6 hours.

Transvaginal sonographic and hysteroscopic findings in postmenopausal women receiving tamoxifen.

STUDY OBJECTIVE: To evaluate the effect of tamoxifen on the endometrium. DESIGN: Prospective study. SETTING: Department of Obstetrics and Gynecology of the University of Rome. PATIENTS: One hundred one postmenopausal women with breast cancer receiving tamoxifen 20 to 30 mg/day for at least 1 year; of these 78 were asymptomatic and 23 had vaginal bleeding. INTERVENTIONS: All patients underwent transvaginal color Doppler sonography. Those with endometrial thickness greater than 5 mm were advised to undergo hysteroscopy and if necessary endometrial biopsy. For women with endometrial thickness less than 5 mm, hysteroscopy was recommended only if irregular endometrial echotexture was observed. MEASUREMENTS AND MAIN RESULTS: Eleven (14%) asymptomatic patients and 1 (4.3%) with vaginal bleeding had endometrial thickness less than 5 mm (p = 0.4, NS). Women with vaginal bleeding had a significantly thicker endometrium than asymptomatic patients (15.8 +/- 7.5 vs 11.1 +/- 5.7 mm, p = 0.003). In the asymptomatic group 31 polyps, 15 atrophic endometria, and 6 hyperplasias were observed. Two endometrial cancers, 13 polyps, and 3 hyperplasias were detected in patients with vaginal bleeding. Hysteroscopy did not always allow endometrial biopsy, even in the presence of increased endometrial thickness with or without irregular surface. No statistical differences were found for mean pulsatility and resistance indexes of uterine and endometrial arteries between symptomatic and asymptomatic women, but these indexes were significantly lower compared with normal postmenopausal values. CONCLUSION: Women receiving tamoxifen, especially those who are asymptomatic, should be closely monitored by transvaginal sonography and hysteroscopy to detect endometrial pathologies.

Differential expression of IGF-I and IGF-II in eutopic and ectopic endometria of women with endometriosis and in women without endometriosis.

PROBLEM: Factors regulating the development, growth, and differentiation of endometrial cells of endometriotic lesions are poorly understood. To investigate the paracrine-autocrine regulation of ectopic endometrial cell growth, the expression of IGF-I and IGF-II were studied. METHOD: Tissue specimens of eutopic and ectopic endometria were obtained from eight patients with endometriosis at laparoscopy and from the endometria of 14 women without endometriosis as controls. They were tested for the expression of IGF-I and IGF-II by immunohistochemical analysis. RESULTS: Immunohistochemical study for IGF-I in controls showed a more intense staining during the proliferative phase both in stromal and epithelial cells. In eutopic endometria of women with endometriosis a reduction in the staining was observed, whereas in epithelial cells of fibrotic peritoneal adhesions an intense immunostaining for IGF-I was observed. Immunohistochemical study of IGF-II in controls showed a more intense staining during secretory phase both in stromal and epithelial cells. In eutopic endometria of women with endometriosis, a reduction in the staining was observed, whereas in epithelial cells of fibrotic peritoneal adhesions an intense immunostaining for IGF-I was observed. Immunohistochemical study of IGF-II in controls showed a more intense staining during secretory phase both in stromal and epithelial cells. In eutopic endometria of women with endometriosis, a reduction in the staining was observed, whereas in epithelial cells of ovarian lesions and fibrotic peritoneal adhesions, no immunostaining for IGF-II was observed. CONCLUSIONS: In endometriosis there is an alteration of mechanisms regulating cell proliferation and differentiation.

Transvaginal Sonographic and Hysteroscopic Findings in Postmenopausal Women Receiving Tamoxifen

The effect of tamoxifen on the endometrium was assessed by transvaginal color Doppler sonography, hyteroscopy, and histology in 90 postmenopausal women, of whom 71 were asymptomatic and 19 had vaginal bleeding. All were treated with tamoxifen 20 to 30 mg/day for breast cancer for at least 1 year and all underwent transvaginal color Doppler sonography. Women with endometrial thickness greater than 5 mm and with vaginal bleeding were advised to undergo hysteroscopy and endometrial biopsy if necessary. In asymptomatic women with endometrial thickness of 5 mm or less, hysteroscopy was recommended if irregular endometrial echotexture was observed. Nine (12.7%) asymptomatic patients and 1 (5.3%) with vaginal bleeding had an endometrial width of 5 mm or less (p = 0.6, NS). Those with vaginal bleeding receiving tamoxifen had a significantly thicker endometrium than asymptomatic women (16.1 ± 7.7 vs 11.1 ± 5.7 mm, p = 0.003). A typical sonographic endometrial pattern with small hypoechoic cystic areas was reported in 83% of patients. In the asymptomatic group, 25 polyps, and 7 atrophic and 13 thickened endometria were observed at hysteroscopy. Two endometrial cancers, 11 polyps, and 1 atrophic and 4 thickened endometria were present in women with vaginal bleeding. Hysteroscopy did not always allow endometrial biopsy even if endometrial thickness with or without an irregular surface was observed. No statistical differences were revealed in the two tamoxifen groups for the mean pulsatility and resistance indexes of the uterine and endometrial arteries, which were otherwise significantly lower compared with normal postmenopausal values. Women receiving tamoxifen, especially asymptomatic women, should be closely monitored by transvaginal color Doppler ultrasonography and hysteroscopy to detect endometrial pathologies.

Vaginal Ultrasonography and Diagnostic Hysteroscopy for Women with Abnormal Uterine Bleeding after Menopause

Three hundred twenty-five postmenopausal women with abnormal uterine bleeding had transvaginal ultrasound examinations. In 158 the endometrial thickness was greater than 5 mm, in 20 less than 5 mm but irregular, and in 147 less than 5 mm and regular. Diagnostic hysteroscopy was performed in the first two groups and showed mild endometrial abnormalities in 23 women, severe in 23, endometrial polyps in 55, myoma in 10, and normal endometrium (atrophic or proliferative) in 22, with synechiae in 5. Forty-six (32%) endometrial biopsies were performed and showed 20 adenocarcinomas, 3 atypical hyperplasias, 10 simple hyperplasias, 5 atrophies, and 8 proliferative. In 18 patients the examination was not possible because of cervical stenosis and was performed under general anesthesia; polyps were removed by operative hysteroscopy. In patients with endometrial thickness greater than 5 mm, hysteroscopy revealed only two cases of mild endometrial abnormalities (cystic atrophy), two polyps, and two myomas. The frequency of endometrial cancer was 7%, similar to that reported by others. Ultrasonography is sensitive in evaluating abnormal uterine bleeding with or without endometrial pathology. Hysteroscopy must be the second procedure because it can exclude pathology and allow a targeted biopsy to confirm the diagnosis.

Costs and Benefits of Office Diagnostic Hysteroscopy

In Italy, the main location for performing diagnostic hysteroscopy is the office. The success of office hysteroscopy is related to savings of time and money and the convenience for the physician and patient. We evaluated 4000 diagnostic hysteroscopies performed between January 2, 1989, and March 1, 1996. In 17.8% of these patients we subsequently performed operative hysteroscopies (metroplasty, synechiolysis, myomectomy, polypectomy for polyps >2 cm), and in 5.2% a total abdominal hysterectomy for abnormal uterine bleeding or endometrial cancer. The low percentage of operations underlines the efficacy of office diagnostic hysteroscopy, especially if we evaluate costs and accuracy of this procedure in detecting uterine pathologies.