Internal controls for quantitative polymerase chain reaction of swine mammary glands during pregnancy and lactation.

High-throughput microarray analysis is an efficient means of obtaining a genome-wide view of transcript profiles across physiological states. However, quantitative PCR (qPCR) remains the chosen method for high-precision mRNA abundance analysis. Essential for reliability of qPCR data is normalization using appropriate internal control genes (ICG), which is now, more than ever before, a fundamental step for accurate gene expression profiling. We mined mammary tissue microarray data on >13,000 genes at -34, -14, 0, 7, 14, 21, and 28 d relative to parturition in 27 crossbred primiparous gilts to identify suitable ICG. Initial analysis revealed TBK1, PCSK2, PTBP1, API5, VAPB, QTRT1, TRIM41, TMEM24, PPP2R5B, and AP1S1 as the most stable genes (sample/reference = 1 +/- 0.2). We also included 9 genes previously identified as ICG in bovine mammary tissue. Gene network analysis of the 19 genes identified AP1S1, API5, MTG1, VAPB, TRIM41, MRPL39, and RPS15A as having no known co-regulation. In addition, UXT and ACTB were added to this list, and mRNA abundance of these 9 genes was measured by qPCR. Expression of all 9 of these genes was decreased markedly during lactation. In a previous study with bovine mammary tissue, mRNA of stably expressed genes decreased during lactation due to a dilution effect brought about by large increases in expression of highly abundant genes. To verify this effect, highly abundant mammary genes such as CSN1S2, SCD, FABP3, and LTF were evaluated by qPCR. The tested ICG had a negative correlation with these genes, demonstrating a dilution effect in the porcine mammary tissue. Gene stability analysis identified API5, VABP, and MRPL39 as the most stable ICG in porcine mammary tissue and indicated that the use of those 3 genes was most appropriate for calculating a normalization factor. Overall, results underscore the importance of proper validation of internal controls for qPCR and highlight the limitations of using absence of time effects as the criteria for selection of appropriate ICG. Further, we showed that use of the same ICG from one organism might not be suitable for qPCR normalization in other species.

Nanotechnologies applied to the analysis of the animal genome.

Whole genome sequence information and high throughput technologies are speeding up the investigation of cellular processes leading to the phenotypic expression of genetic information. Nanotechnologies provide innovative tools to accomplish this task, increasing throughput and sensitivity and decreasing cost and time of analyses. Goals as ambitious as the sequencing of a mammalian-sized genome in a matter of hours, and of detecting gene expression from a single cell, are just around the corner. Animal breeding will benefit from these advances in the understanding of the genetic basis of complex traits and in the application of molecular information for marker and gene assisted selection.

Phase II study of cisplatin and vinorelbine as first-line chemotherapy in patients with carcinoma of the uterine cervix.

PURPOSE: To evaluate the activity and toxicity of the combination of cisplatin (80 mg/m2 day 1) and vinorelbine (25 mg/m2 days 1 and 8) in patients with carcinoma of the uterine cervix that has not been previously treated with chemotherapy. PATIENTS AND METHODS: Fifty patients with cervical cancer were enrolled onto this study (27 stage IB-III, 23 stage IVB-recurrent). A two-stage optimal Simon design was applied. Thirteen responders of 29 treated patients were required to proceed beyond the first stage, and 28 responders were needed overall. RESULTS: Hematologic toxicity was mild, with neutropenia being the most frequent side effect. Nonhematologic toxicity was frequent but never severe; one patient had grade 3 peripheral neurotoxicity. Objective responses were recorded for 32 patients (64%): 11 patients (22%) achieved a complete response (CR) and 21 patients (42%) achieved a partial response (PR). The response rate was 81.5% in patients with IB-III stage (25.9% CR rate) and 43.5% in patients with IVB-recurrent disease (17.4% CR rate). Responses were seen both in stage IVB patients (one CR and two PRs, for an overall rate of 37.5%) and in patients with recurrent disease (three CRs + four PRs, for an overall rate of 46.7%). CONCLUSION: The combination of cisplatin and vinorelbine is an active regimen in the treatment of patients with early-stage and advanced carcinoma of the uterine cervix. The hematologic and nonhematologic toxicity of this combination is mild.

Carboplatin, cisplatin and paclitaxel in the treatment of patients with epithelial ovarian cancer.

BACKGROUND: Prognosis of advanced ovarian cancer is unsatisfactory. Chemotherapy can be intensified combining active drugs at their highest possible doses. PATIENTS AND METHODS: In this phase I/II trial, 77 untreated patients received escalating doses of paclitaxel (135, 155, 175, 195 and 215 mg/m2, infused over 3 hours) with carboplatin (AUC 3.6) and cisplatin (60 mg/m2). Nine, 16, 13, 8 and 3 patients were treated at the five levels, respectively. A further 28 patients were treated at the maximum tolerable dose (MTD). RESULTS: Dose-limiting toxicities (one WHO grade 3 constipation, one grade 2 prolonged peripheral neurotoxicity and one grade 3 cardiac toxicity) occurred at 215 mg/m2 in 3 out of 3 patients. MTD was reached at level 4 paclitaxel dose (195 mg/m2). Response was evaluated in 62 patients. A complete response was achieved in 23 patients (37.1%-95% CI 25.2-50.3), including 16 (25.8%) pathological and partial response in 28 (45.2%), for an overall response rate of 82.3% (95% exact CL: 70.5%-90.8%). The probability of response was affected by the degree of initial debulking (p = 0.002) and not by the paclitaxel dose. In patients with stage III-IV disease, median progression-free survival was 17 months (95% CI 14-25). After a median follow-up of 28 months, median survival had not been reached; 2-year estimated survival was 67%. CONCLUSION: Paclitaxel can be safely given at the dose of 195 mg/m2 in combination with carboplatin (AUC 3.6) and cisplatin (60 mg/m2). This combination is active and safe and could be considered in clinical settings requiring intensive short treatment.

Primary malignant melanoma of the vagina: case report.

Primary vaginal melanoma is a very rare gynecological malignant tumor (less than 150 reported cases to-date). Prognosis is poor in spite of treatment. Due to the fact that only small groups of patients have been compared, conservative treatment has usually been recommended. In recent times, radical pelvic surgery has appeared to improve the chances of survival. We present an unusual case of primitive melanoma of the upper-third of the vagina with urethral and urinary bladder infiltration in a 47-year-old woman. Treatment consisted of preliminary pelvic bilateral lymphadenectomy, anterior exenteration, and urinary bladder reconstruction according to the Bricker technique. Four months after surgical treatment, liver metastases were found. Chemotherapy was initiated consisting of 8 cycles every 21 days of fotemustine 100 mg/m2 (day 1) and dacarbazine (DTIC) 250 mg/m2 (days 2-5). Interferon alpha-2-b, 3 MU thrice weekly, for the whole period of chemotherapy, was also administered. The patient is in partial remission one year after surgical treatment.

From endothelial dysfunction to atherosclerosis.

It has recently emerged that endothelial dysfunction is an early step in the development of atherosclerosis and is mainly characterised by a reduction in the bioavailability of nitric oxide. All of the traditional cardiovascular (CV) risk factors (dyslipidemia, arterial hypertension, hyperglycemia and diabetes) are associated with endothelial dysfunction, and oxidised low-density lipoproteins, the renin-angiotensin axis and insulin resistance play important roles in the pathogenesis of impaired endothelial function. The increased expression of adhesion molecules and pro-inflammatory cytokines leads to abnormal endothelium-dependent vasodilation which could be investigated using vasoreactivity tests such as flow-mediated dilation in the brachial artery. Recently, new evidences showed that the immune system plays an important role in the pathogenesis of endothelial dysfunction and atherosclerosis with a particular regard towards autoimmunity. The high prevalence of the atherosclerotic process in systemic autoimmune diseases supports the hypothesis of the immune pathogenesis. Evaluating coronary microvascular dysfunction by means of transthoracic echocardiography with non-invasive coronary flow reserve assessment is particularly interesting as it could detect preclinical impairment of coronary microvascular function. The discovery that the mechanisms responsible for endothelial damage have a genetic basis could improve the approach to CV diseases. This review summarises the most important aspects of the pathogenesis and development of endothelial dysfunction, with particular attention to the role of traditional CV risk factors, the usefulness of vasoreactivity tests, and the future perspectives opened by genetic studies.