FAM19A4/miR124-2 methylation analysis as a triage test for HPV-positive women: cross-sectional and longitudinal data from a Dutch screening cohort

ObjectivesThe aim was to evaluate the cross-sectional and long-term triage performance of FAM19A4/miR124-2 methylation analysis in human papillomavirus (HPV)-based cervical screening.MethodsWe conducted a post hoc analysis within a Dutch population-based HPV-positive study cohort of women aged 30–60 years (n = 979). Cross-sectional cervical intraepithelial neoplasia (CIN) 3+ sensitivity, specificity, positive predictive value and negative predictive value as well as cumulative CIN3+ or cervical cancer risks after 9 and 14 years were compared for three baseline triage strategies: (1) cytology, (2) FAM19A4/miR124-2 methylation analysis and (3) combined FAM19A4/miR124-2 methylation with cytology.ResultsCIN3+ sensitivity of FAM19A4/miR124-2 methylation analysis was similar to that of cytology (71.3% vs 76.0%, ratio 0.94, 95% CI 0.84 to 1.05), at a lower specificity (78.3% vs 87.0%, ratio 0.90, 95% CI 0.86 to 0.94). Combining FAM19A4/miR124-2 methylation analysis with cytology resulted in a CIN3+ sensitivity of 84.6% (95% CI 78.3 to 90.8) at a specificity of 69.6% (95% CI 66.5 to 72.7). Similar 9- and 14-year CIN3+ risks for baseline cytology-negative women and baseline FAM19A4/miR124-2 methylation-negative women were observed, with risk differences of –0.42% (95% CI –2.1 to 1.4) and –0.07% (95% CI –1.9 to 1.9), respectively. The 14-year cumulative cervical cancer incidence was significantly lower for methylation-negative women compared to cytology-negative women (risk difference 0.98%, 95% CI 0.26 to 2.0).DiscussionFAM19A4/miR124-2 methylation analysis has a good triage performance on baseline screening samples, with a cross-sectional CIN3+ sensitivity and long-term triage-negative CIN3+ risk equalling cytology triage. Therefore, FAM19A4/miR124-2 methylation analysis appears to be a good and objective alternative to cytology in triage scenarios in HPV-based cervical screening.     

DNA methylation testing for endometrial cancer detection in urine,cervicovaginal self-samples and cervical scrapes

Endometrial cancer incidence is rising and current diagnostics often require invasive biopsy procedures. DNA methylation marker analysis of minimally- and non-invasive sample types could provide an easy-to-apply and patient-friendly alternative to determine cancer risk. Here, we compared the performance of DNA methylation markers to detect endometrial cancer in urine, cervicovaginal self-samples and clinician-taken cervical scrapes. Paired samples were collected from 103 patients diagnosed with stage I to IV endometrial cancer. Urine and self-samples were collected at home. All samples were tested for nine DNA methylation markers using quantitative methylation-specific PCR. Methylation levels measured in endometrial cancer patients were compared to unpaired samples of 317 healthy controls. Diagnostic performances were evaluated by univariable and multivariable logistic regression analysis, followed by leave-one-out cross-validation. Each methylation marker showed significantly higher methylation levels in all sample types of endometrial cancer patients compared to healthy controls (P < .01). Optimal three-marker combinations demonstrated excellent diagnostic performances with area under the receiver operating curve values of 0.95 (95% CI: 0.92-0.98), 0.94 (0.90-0.97) and 0.97 (0.96-0.99), for endometrial cancer detection in urine, self-samples and scrapes, respectively. Sensitivities ranged from 89% to 93% at specificities of 90% to 92%. Virtually equal performances were obtained after cross-validation and excellent diagnostic performances were maintained for stage I endometrial cancer detection. Our study shows the value of methylation analysis in patient-friendly sample types for endometrial cancer detection of all stages. This approach has great potential to screen patient populations at risk for endometrial cancer.     

Theophylline improves hypoglycemia unawareness in type 1 diabetes

Iatrogenic hypoglycemias and the subsequent occurrence of hypoglycemia unawareness are well-known complications of intensive insulin therapy in type 1 diabetic patients that limit glycemic management. From a pharmacological point of view, the adenosine-receptor antagonist theophylline might be beneficial in the management of hypoglycemia unawareness. Theophylline stimulates the release of catecholamines and reduces cerebral blood flow, thereby facilitating stronger metabolic responses to and a prompter perception of decreasing glucose levels. To test the effect of theophylline on responses to hypoglycemia, we performed paired hyperinsulinemic-hypoglycemic clamp studies in 15 diabetic patients with hypoglycemia unawareness and 15 matched healthy control subjects. In random order, we concurrently infused either theophylline or placebo. Measurements included counterregulatory hormones, symptoms, hemodynamic parameters, and sweat detection using a dew-point electrode. Additionally, middle cerebral artery velocities (V(MCA)) using transcranial Doppler were monitored as an estimate of cerebral blood flow. When compared with placebo, theophylline significantly enhanced responses of plasma epinephrine, norepinephrine, and cortisol levels in both diabetic patients and control subjects. Because of the theophylline, sweat production started at approximately 0.3 mmol/l higher glucose levels in both groups (P < 0.01), and symptom scores in diabetic patients approached those in control subjects. Theophylline decreased V(MCA) in both groups (P < 0.001), but significantly greater in diabetic patients (P < 0.01), and prevented the hypoglycemia-induced increase of V(MCA) that occurred during the placebo studies. We conclude that theophylline improves counterregulatory responses to and perception of hypoglycemia in diabetic patients with impaired awareness of hypoglycemia.     

Intra-population plasticity of Anopheles darlingi's (Diptera, Culicidae) biting activity patterns in the state of Amapá, Brazil

OBJECTIVE: To assess the variation in Anopheles darlingi's biting activity compared to An. marajoara in the same locality and to biting activity data from other regions. METHODS: Using human bait, eight observations of the biting activity of An. darlingi and An. marajoara were carried out during 1999 and 2000 in the municipality of S?o Raimundo do Pirativa, state of Amapá, Brazil. Each observation consisted of three consecutive 13-hour collections, close to full moon. There were shifts of collectors in the observation points and nocturnal periods. RESULTS: An. darlingi revealed considerable plasticity of biting activity in contrast to An. marajoara, which showed well-defined crepuscular biting peaks. No significant correlation between density and biting activity was found, but a significant correlation existed between time and proportional crepuscular activity, indicating underlying ecological processes not yet understood. Two of the four available data sets having multiple observations at one locality showed considerable plasticity of this species' biting patterns as well. CONCLUSION: Intra-population variation of biting activity can be as significant as inter-population variation. Some implications in malaria vector control and specific studies are also discussed.     

Alternative acetylcholinesterase molecular forms exhibit similar ability to induce neurite outgrowth

Several groups have reported that acetylcholinesterase (AChE), through a mechanism not involving its catalytic activity, may have a role in fiber elongation. These observations were performed on experimental systems in which acetylcholine synthesis was active. Because neurite outgrowth can be modulated by neurotransmitters, we used the N18TG2 neuroblastoma line, which is defective for neurotransmitter production, to evaluate whether AChE may modulate neurite sprouting in nonenzymatic ways. To avoid the possibility that differences between transfected and mock-transfected clones may be due to the selection procedure, N18TG2 cells were previously subcloned, and the FB5 subclone was used for transfections. We performed transfections of FB5 cells with three distinct constructs encoding for the glycosylphosphoinositol-anchored AChE form, the tetrameric AChE form, and a soluble monomeric AChE form truncated in its C-terminus. A morphometric analysis of retinoic acid-differentiated clones was also undertaken. The results revealed that higher AChE expression following transfection brings about a greater ability of the clones to grow fibers with respect to nontransfected or mock-transfected cells irrespective of the used construct. Having observed no differences between the morphology of the transfected clones, we tested the possibility that the culture substrate can affect the capability of the clones to extend fibers. Also in this case we revealed no differences between the clones cultured on uncoated or collagen-pretreated dishes. These data indicate that alternative AChE molecular forms that differ in their C-teminal region exhibit similar ability to induce fiber outgrowth and suggest that the protein region responsible for this role is located in the invariant portion of the AChE molecule.     

Value and cost of follow-up after adjuvant treatment of patients with Dukes' C colonic cancer

BACKGROUND: The clinical value and costs of different diagnostic tools used to identify potentially curable recurrent disease in patients treated adjuvantly for curatively resected Dukes' C colonic cancer were examined. METHODS: The study group comprised 496 patients treated with chemotherapy over a 1-year interval. Follow-up consisted of interim history, physical examination, liver ultrasonography or computed tomography (CT), measurement of carcinoembryonic antigen (CEA) levels, chest radiography and colonoscopy. RESULTS: Two hundred and thirteen patients had recurrent disease (median follow-up 43 months). Forty-two patients with recurrence (20 per cent) were treated with curative intent (median survival 38 months; 5-year survival rate 40 per cent). Recurrence was identified by liver ultrasonography or CT (n = 14), evaluation of symptoms (n = 12), colonoscopy (n = 8), CEA measurement (n = 3), chest radiography (n = 2), physical examination (n = 1) and other modalities in two patients. The mean cost of diagnostic procedures per curative resected recurrence for patients amenable to salvage surgery was US$9011. Of all treatable recurrences, 12 of 42 were identified by evaluation of symptoms only. Ultrasonography and colonoscopy identified 22 recurrences at a cost of US$11 790 per patient, while routine follow-up by CEA measurement, chest radiography and physical examination identified a further six at a cost of US$19 850 per patient. CONCLUSION: Potentially curable recurrences were detected primarily by liver imaging and colonoscopy. The yield of CEA measurement, chest radiography and physical examination was relatively low; such methods were expensive and should not be recommended in the routine follow-up of these patients.     

Differences between rabbit sinoatrial pacemakers in their response to Mg, Ca and temperature

The rabbit sinoatrial node is functionally inhomogeneous with respect to its response to changes in Mg concentration (0.6 to 6.0 mmol X litre-1) and in Ca concentration (1.1 to 2.2 mmol X litre-1) and to changes in experimental temperature (30 to 38 degrees C). High Mg (6.0 mmol X litre-1) stabilises the position of the leading pacemaker. This pacemaker decelerates under high Mg, but the subsidiary ones decelerate even more. Consequently when a subsidiary pacemaker turns dominant--eg under low Ca or at low temperature--an enhanced chronotropic response to high Mg is observed. The superior (cranial) part of the rabbit sinoatrial node is more responsive to changes in Ca concentration than the inferior (caudal) part. The same holds true for changes in temperature.     

Sinus node and atrium cells from the rabbit heart: a quantitative electron microscopic description after electrophysiological localization.

Electrophysiologically identified cell groups in the sinus node from the rabbit have been compared with atrial fibers with the electron microscope. The point counting method has been used to estimate the volume density of the following structures: nucleus, mitochondria, myofilaments, sarcoplasmic reticulum tubules and subsarcolemmal vesicles. These data were collected in leading pacemaker cells, latent pacemaker cells and atrium cells from the crista terminalis.It has been found that organized structures in leading pacemaker cells occupy about 50% of the cell volume, as compared with over 90% in atrial fibers. Leading pacemaker cells consequently appear extremely “empty”. It has also been found that the group of cells which show the characteristic features of leading pacemaker cells at the ultrastructural level as observed in the correlated experiments is larger than the leading center found in electrophysiology and thus it seems impossible with the actual observation methods to delineate the leading pacemaker center using only cytological criteria.