NOTCH1 and PIK3CA mutation are related to HPV-associated vulvar squamous cell carcinoma.

NOTCH1 and PIK3CA are members of important cell signalling pathways that are deregulated in squamous cell carcinomas of various organs. Vulvar squamous cell carcinomas (vulvSCC) are classically divided into two pathways, HPV-associated or HPV-independent, but the effect of NOTCH1 and PIK3CA mutations in both groups is unclear. We analysed two different cohorts of vulvSCC using Hybrid Capture-based Comprehensive Genomic Profiling and identified NOTCH1 and PIK3CA mutations in 35% and 31% of 48 primary vulvSCC. In this first cohort, PIK3CA and NOTCH1 mutations were significantly correlated with HPV infection (p < 0.01). Furthermore, mutations in both genes were associated with an advanced tumor stage and poorly differentiated status (p < 0.05). PIK3CA and NOTCH1 mutations were also associated with shorter patient survival which did not reach significance. In the second cohort of 735 advanced vulvSCC from metastatic site biopsies or from sites of unresectable loco-regional disease, NOTCH1 and PIK3CA mutations were reported in 14% and 20.3%, respectively. 4 of 48 (8%) and 22 of 735 vulvSCC (3.0%) featured genomic alterations (short variants and/or copy number changes and/or rearrangements) in both NOTCH1 and PIK3CA. NOTCH1 mutations were mostly located in the extracellular EGF-like domains, were inactivating and indicated that NOTCH1 functions predominantly as a tumor suppressor gene in vulvSCC. In contrast, PIK3CA mutations favored hotspot codons 1624 and 1633 of the gene, indicating that PIK3CA acts as an oncogene in vulvar carcinogenesis. In conclusion, NOTCH1 and PIK3CA mutations are detectable in a substantial proportion of vulvSCC and are related to HPV infection and more aggressive tumor behaviour.

PD-L1 receptor expression in vulvar carcinomas is HPV-independent.

PD-L1 (programmed cell death 1 ligand) is expressed on many cancer cells and prevents tumor cell death by blocking T cell activity. PD-L1 overexpression has been reported in squamous cell carcinomas of head and neck and lung cancer. To better understand the role of PD-L1 expression in vulvar cancer, we analyzed PD-L1 expression by immunohistochemistry in 55 well-characterized squamous cell carcinomas of vulva. PD-L1 was found in 72.7% of tumors. 27.3% of vulvar carcinomas showed moderate or strong PD-L1 expression. PD-L1 expression was correlated with low tumor stage (p < 0.05). There was no association to other clinicopathological parameters, HPV status, and overall survival of vulvar carcinoma patients. In conclusion, PD-L1 overexpression is detectable in a substantial proportion of vulvar carcinomas in all stages independent of HPV and may be a suitable therapeutic target in these cancers.

[Assessment of mental states at risk of psychotic transition: validation of the French version of the CAARMS]. / Évaluation des états mentaux à risque de transition psychotique: validation de la version française de la CAARMS.

This article aims to present the validation study of the French version of the Comprehensive Assessment of at risk mental states (CAARMS), an interview that seeks to determine whether young adults criteria for at-risk (AR) mental states, or psychosis. We assessed 40 young subjects, 15 were considered as "prodromal" (Prd) and 10 as experiencing a first episode of psychosis (PEP) by our expert clinician at the center - centre d'évaluation des jeunes adultes et adolescents, University Hospital Centre, Paris - and 15 were healthy controls matched for age and sex. When assessed with the CAARMS, 73 % (n=11) of the prodromal subjects reached the criteria for AR mental state, four subjects did not reach the criteria for AR, nor psychosis (P) and 100 % of the PEP reached the criteria for P. The three groups were significantly different on CAARMS total score (P<0.001) and subscores ; Prd subjects had intermediate scores between PEP (P<0.001) and controls (P<0.001) scores, PEP showing the highest scores. Post-hoc analysis showed that Prd significantly differed from Controls on each subscale (P<0.001) and that Prd differed from PEP on the "positive symptoms" subscale (P<0.001), as well as on "behavioural change" (P=0.021), owing to difference on the item "impaired role function". We used the brief psychiatric rating scale 24 items with anchor (BPRS24-EA) in addition to with the CAARMS, the AR group showed intermediate scores between controls and P subjects. Total scores of both scales were correlated (r=0.408 ; P=0.043) and the BPRS24-EA "positive symptoms" score was correlated with CAARMS' scores on the "Positive symptoms" subscale (r=0.456, P=0.022), "emotional disturbance" (r=0.506, P=0.01), and "behavioural change" (r=0.666 P=0.001). We found no correlation between BPRS negative and depression subscales and any of the CAARMS' subscales. When looking at its reliability, reliability coefficients (Cronbach's alpha) showed excellent reliability for "positive symptoms", "emotional disturbance", "behavioural change" and "general psychopathology" (respectively r=0.82, 0.75, 0.78, 0.84, 0.83) and moderate reliability for "cognitive change", "negative symptoms" and "motor/physical change" (respectively r=0.39, 0.59, 0.43). Overall, analysis of the results of construct validity, concurrent validity and reliability of the CAARMS indicates that the French version is valid and reliable. It is now available to develop and implement early detection programs in French speaking countries.

A pilot study of methyl chloride emissions from tropical woodrot fungi.

Flux chamber measurements made in a rainforest provide evidence that methyl chloride is emitted from rotting wood. However, its net flux was found to be into the soil, probably due to competing production and consumption processes within the soil. Evidence was found for a regional source, possibly vegetation, since its concentration above the canopy was substantially greater than reported average equatorial values.

Influence of pantoprazole on oesophageal motility, and bile and acid reflux in patients with oesophagitis.

BACKGROUND: Reflux of duodeno-gastric juice into the oesophagus appears to be involved in the pathogenesis of both reflux oesophagitis and oesophageal adenocarcinoma. Although proton pump inhibitors have been shown to decrease acid reflux and heal oesophagitis, their effect on biliary reflux and motility is less clear. AIM: To investigate whether pantoprazole also reduces bile reflux and whether this is paralleled by a change in oesophageal motility. METHODS: Combined 24-h measurements of intraoesophageal bilirubin concentration, pH and pressure were performed in 18 symptomatic patients with endoscopically proven reflux oesophagitis before and on day 28 of treatment with pantoprazole, 40 mg/day, under standardized conditions. A reflux symptom score was determined initially and every 2 weeks thereafter. After 56 days on medication, a control endoscopy was performed. RESULTS: The symptom score and the acid and bile reflux improved significantly, whereas the motility parameters did not change during the study period. Helicobacter pylori-positive patients had a significantly higher bile reflux time (32.1 +/- 4.3%) than H. pylori-negative patients (16.3 +/- 3.1%) (P=0.009). The endoscopic healing rate was 89%. The cough symptoms disappeared in three of four patients. CONCLUSIONS: The proton pump inhibitor pantoprazole decreases both acid and bile reflux. The decrease of bile reflux cannot be explained by increased oesophageal clearance as oesophageal motility did not improve with therapy. Interestingly, H. pylori infection of the stomach was associated with higher levels of oesophageal bile reflux.

Expression and localisation of synaptotagmin isoforms in endocrine beta-cells: their function in insulin exocytosis.

Exocytosis of insulin containing Large Dense Core Vesicles (LDCVs) from pancreatic beta-cells and derived cell lines is mainly controlled by Ca(2+). Several lines of evidence have demonstrated a role of the Ca(2+)- and phospholipid-binding protein synaptotagmin (syt) in this event. Synaptotagmins form a large protein family with distinct affinities for Ca(2+) determined by their two C(2) domains (C(2)A/B). Except for the well-characterized isoforms I and II, their role is still unclear. We have used here insulin-secreting cells as a model system for LDCV exocytosis to gain insight into the function of synaptotagmins. Immunocytochemical analysis revealed that of the candidate Ca(2+) sensors in LDCV exocytosis, syt III was not expressed in primary beta-cells, whereas syt IV was only found adjacent to the TGN. However, syt V-VIII isoforms were expressed at different levels in various insulin-secreting cells and in pancreatic islet preparations. In streptolysin-O permeabilized primary beta-cells the introduction of recombinant peptides (100 nM) corresponding to the C(2) domains of syt V, VII and VIII, but not of syt III, IV or VI, inhibited Ca(2+)-evoked insulin exocytosis by 30% without altering GTP gamma S-induced release. Our observations demonstrate that syt III and IV are not involved in the exocytosis of LDCVs from primary beta-cells whereas V, VII and VIII may mediate Ca(2+)-regulation of exocytosis.

Five-year audit of ambulatory 24-hour esophageal pH-manometry in clinical practice.

BACKGROUND: Esophageal function testing was developed to aid diagnosis in patients with negative endoscopy. Although combined 24-h esophageal pH-manometry is now commercially available, its routine clinical effectiveness has not yet been studied. METHODS: From 1992 to 1996 we evaluated 303 consecutive patients who were first-time referrals to our unit for 24-h esophageal pH-manometry. The referral indications were gastroesophageal reflux disease, 47.2%; dysphagia, 18.5%; non-cardiac chest pain, 14.9%; connective tissue disease, 13.2%; and symptomatic patients after antireflux surgery, 6.3%. RESULTS: Overall, esophageal function testing altered the diagnosis of 44% of the patients, confirmed it in 38%, and specifically changed the management of 66%. The final clinical 'diagnosis' was reflux disease, 54% (32% with non-specific esophageal motility disorder); connective tissue disease, 9.9%; achalasia, 9.6%; other specific esophageal motility disorders, 3.3%; non-specific esophageal motility disorders, 6.9%; and normal, 16.2%. The cost per testing was estimated to be US$305 and per change in management US$465. CONCLUSION: Combined 24-h pH-manometry has been shown to be a useful and cost-effective test for the management of selected patients in whom the primary investigation was insufficient.

Autopsy: quality assurance in the ICU.

OBJECTIVE: To examine the correlation between the clinical diagnosis and autopsy findings in adult patients who died in an intensive care unit (ICU). To determine the rate of agreement of the basic and terminal causes of death and the types of errors in order to improve quality control of future care. DESIGN: Retrospective study. SETTING: Adult ICU in a university hospital. PATIENTS: 30 adult patients who died in the ICU, with the exclusion of medicolegal cases. METHODS AND MAIN RESULTS: Anatomo-clinical meetings were held to analyze the pre- and postmortem correlations in 30 consecutive autopsies at the ICU of the University Hospital, School of Medicine of Botucatu/ UNESP, from January 1994 to January 1997. The rate of correct clinical diagnoses of the basic cause was 66.7%; in 23.3% of cases, if the correct diagnosis was made, management would have been different, as would have been the evolution of the patient's course (Class I error); in 10% of the cases the error would not have led to a change in management (Class II error). The rate of correct clinical diagnoses of terminal cause was 80%. CONCLUSIONS: The rate of recognition of the basic cause was 66.7%, which is consistent with the literature, but the Class I error rate was higher than that reported in the literature.

Effect of repeated injection and continuous infusion of omeprazole and ranitidine on intragastric pH over 72 hours.

OBJECTIVE: In healthy subjects and patients with bleeding peptic ulcers, ranitidine and omeprazole, given parenterally, achieve high intragastric pH values on the first day of therapy. However, data on the antisecretory effect beyond the first 24 h is scanty. In addition, the superiority of either infusion or injection of omeprazole remains unproven. Thus, we have compared the antisecretory effect of high dose omeprazole and ranitidine infusion and injection over the critical first 72 h. METHODS: A total of 34 healthy volunteers were randomized into a double-blind crossover 72 h intragastric pH-metry study (data compared: median pH, percentage of time with pH >4 and pH >6). Omeprazole-infusion: initial bolus of 80 mg + 8 mg/h; omeprazole-injection: initial bolus of 80 mg + 40 mg/6 h; Ranitidine-infusion: initial bolus of 50 mg + 0.25 mg/kg/h; ranitidine-injection: 100 mg/6 h. RESULTS: Omeprazole-infusion versus ranitidine-infusion: on day 1: median pH 6.1 vs 5.1 (p = 0.01) and 95% vs 70% was pH >4 (p < 0.01); on day 2: median pH 6.2 vs 3.2 (p < 0.01); and 100% vs 38% was pH >4 (p < 0.01); on day 3: median pH 6.3 vs 2.7 (p < 0.01); 100% vs 26% was pH >4 (p < 0.01). Injections of both drugs were significantly less effective than the infusions on day 1. Thereafter, omeprazole injection was almost as effective as omeprazole infusion, whereas ranitidine injection and infusion were equally effective. CONCLUSION: Our study shows, for the first time, that omeprazole infusion was significantly superior to all other regimens by having a high median pH >6 on each day. The tolerance effect of ranitidine, however, led to a rapid loss of antisecretory activity on days 2 and 3, rendering it inappropriate for situations in which high intragastric pH-levels appear to be essential.

The cytoplasmic domains of a beta1 integrin mediate polarization in Madin-Darby canine kidney cells by selective basolateral stabilization.

In Madin-Darby canine kidney cells, newly synthesized apical and basolateral membrane proteins are generally transported directly to their respective cell surface domain due to targeting determinants that mediate sorting in the Golgi complex. In several basolateral membrane proteins, these targeting determinants reside in the cytoplasmic domains. We show here that basolateral expression of the human alpha5beta1 integrin in stably transfected Madin-Darby canine kidney cells is also mediated by the cytoplasmic domains. Distinct regions in both cytoplasmic domains were found to be sufficient to mediate basolateral expression independently from one another. Unexpectedly, newly synthesized wild-type alpha5beta1 and basolaterally expressed chimeras containing the cytoplasmic domain of either alpha5 or beta1 were integrated into both cell surface domains, preferentially apically, during biosynthesis. The apical pools of wild-type integrin and chimeric subunits were found to become quickly degraded, whereas the basolateral pools were stabilized. Thus, the cytoplasmic domains of the alpha5beta1 integrin are independently sufficient to mediate sorting by selective basolateral stabilization.

Carbohydrate-mediated Golgi to cell surface transport and apical targeting of membrane proteins.

Polarized expression of most epithelial plasma membrane proteins is achieved by selective transport from the Golgi apparatus or from endosomes to a specific cell surface domain. In Madin-Darby canine kidney (MDCK) cells, basolateral sorting generally depends on distinct cytoplasmic targeting determinants. Inactivation of these signals often resulted in apical expression, suggesting that apical transport of transmembrane proteins occurs either by default or is mediated by widely distributed characteristics of membrane glycoproteins. We tested the hypothesis of N-linked carbohydrates acting as apical targeting signals using three different membrane proteins. The first two are normally not glycosylated and the third one is a glycoprotein. In all three cases, N-linked carbohydrates were clearly able to mediate apical targeting and transport. Cell surface transport of proteins containing cytoplasmic basolateral targeting determinants was not significantly affected by N-linked sugars. In the absence of glycosylation and a basolateral sorting signal, the reporter proteins accumulated in the Golgi complex of MDCK as well as CHO cells, indicating that efficient transport from the Golgi apparatus to the cell surface is signal-mediated in polarized and non-polarized cells.

[Nonsteroidal antirheumatic drugs and acetylsalicylic acid: adverse effects distal to the duodenum]. / Nichtsteroidale Antirheumatika und Azetylsalizylsäure: Nebenwirkungen distal des Duodenums.

An increasing number of case reports and controlled trials have drawn attention to NSAID-induced side effects in the lower gastrointestinal tract. In this review we also report 9 cases of colonic ulcers and 7 cases of diaphragm disease of the ascending colon, most of them associated with the long-term intake of slow release diclofenac. NSAIDs not only can exacerbate preexisting conditions such as inflammatory bowel disease or diverticular disease, but may also induce de novo enteropathy, colitis, collagenous colitis ulcers and strictures. Complications such as bleeding, perforation or bowel obstruction may require surgery. From the literature and our own experience we conclude that the use of slow release formulations has shifted the toxicity of NSAIDs from the upper to the lower gastrointestinal tract. This must be considered in differential diagnosis and checked by endoscopy if appropriate.

Micronutrient antioxidant status in black South Africans with chronic pancreatitis: opportunity for prophylaxis.

Biochemical assessments of micronutrient antioxidant status were done in 14 consecutive black patients with calcific chronic pancreatitis and 15 controls at Soweto, near Johannesburg in southern Africa. The patients showed subnormal levels of vitamin C in plasma; selenium, beta-carotene and alpha-tocopherol in serum; and inorganic sulphate (as an index of long-term sulphur amino acid intake) in urine (P < 0.001 for each): furthermore, among the patients ascorbate constituted a lower fraction of vitamin C (P < 0.002), indicating heightened oxidation of the bioactive form. By comparing the results in Sowetan controls with reference ranges from Manchester, UK, the markedly lower vitamin C and, hence, ascorbate levels in the Sowetans was underlined (P < 0.001) and their selenium levels were also lower (P < 0.001), but beta-carotene, alpha-tocopherol and inorganic sulphate levels were comparable. The very low bioavailability of ascorbate among Sowetan controls is reminiscent of our previous finding in outwardly healthy people at Madras in southern India: in both these areas chronic pancreatitis is currently endemic, has a propensity to pancreatic calculi and runs a virulent course towards premature death from diabetes, malnutrition or pancreatic cancer. Considering that low ascorbate levels are a feature in patients with chronic pancreatitis who develop pancreatic calculi at Manchester and that antioxidant supplements ameliorate painful symptoms, we suggest that poor antioxidant intake may predispose underprivileged tropical communities to the disease. If so, there could be an opportunity for prophylaxis through a daily tablet containing vitamin C, perhaps along with selenium at Soweto and beta-carotene at Madras.

Evidence of toxic metabolite stress in black South Africans with chronic pancreatitis.

We report the results of a further study to test our hypothesis that toxic metabolite stress is germane to heightened free radical activity and hence to the genesis of chronic pancreatitis. Consecutive black South African patients with clinically quiescent chronic pancreatitis were studied, provided that the diagnosis had been made within the previous 2 years and that they did not have overt liver disease. All of them had been advised to stop drinking alcohol. Analysis of an early morning sample of urine showed a lower ratio of inorganic to ester sulphate (P < 0.001) and a higher ratio of D-glucaric acid to creatinine (P < 0.02) in the group of 14 patients than in 15 local controls, while plasma analysis showed a lower concentration of glutathione (GSH) in the patients (P < 0.001). This evidence of increased utilisation of phase II conjugative pathways of xenobiotic disposal was in keeping with on-going toxic metabolite stress from heightened phase I oxidative metabolism in the group of patients. Parallel studies of theophylline pharmacokinetics showed heightened drug clearance compatible with induced cytochrome P-4501A2 in two patients, whereas increased activity of gamma-glutamyl transferase in serum suggested persisting induction of P-4502E1, as by ethanol, in several others. The contemporaneous increases in free radical activity and utilisation of xenobiotic disposal pathways in Sowetan Africans with chronic pancreatitis is in line with the toxic metabolite concept of disease pathogenesis.

A preliminary report on urinary BT-PABA/PAS excretion index, serum pancreatic isoamylase and faecal chymotrypsin tests of pancreatic dysfunction in Sowetan Africans.

The steady increase in chronic pancreatitis among black Africans at Soweto, RSA, in the past 40 years necessitates an objective and non-invasive test to detect the disease at an early stage. Given the biphasic nature of the disease--secretory hyperfunction with periodic active inflammatory episodes followed by steady exocrine impairment--we assessed three potential aids. Urinary BT-PABA/PAS excretion index (PEI), serum pancreatic isoamylase (PIA) and faecal chymotrypsin activity (FCA) were measured in the following groups: 16 outwardly healthy hospital workers, 16 consecutive patients with calcifying chronic pancreatitis and 19 with abdominal pain ascribed to other conditions (disease controls). (1) Healthy controls had lower PEI than those at Manchester, UK, or Madras, India, from subclinical acinar loss--as shown by lower PABA recovery whereas intestinal absorptive capacity was maintained, as shown by recovery of PAS. (2) Using the popular cut-off for PEI (0.75) only 9 of 14 patients with chronic pancreatitis were identified (sensitivity 64%, 2 tests unsatisfactory), while a value of less than 0.54, the mean -2 S.D. in local controls, yielded sensitivity of 50%. (3) If PEI of less than 0.75 or PIA outside the reference range was taken to indicate the disease, 5 of 9 disease controls would have been classed as chronic pancreatitis (among those with both tests satisfactory): retrospective ultrasound scans did not identify these. (4) Although FCA was less than the preselected cut-off, 5 units/g, in every patient with chronic pancreatitis (100% sensitivity) its poor predictive value was indicated by low specificity: subnormal levels in 4 of 14 and 6 of 16 healthy controls or disease controls, respectively, most of whom had near-normal values of PEI, PIA or both. (5) Collectively, these results suggest a high frequency of subclinical chronic pancreatitis at Soweto, but also that the combination of tests required to identify it may prove impractical--whether in field surveys or hospital practice.

Heightened free radical activity in blacks with chronic pancreatitis at Johannesburg, South Africa.

Four indices of free radical activity were measured in fasting serum/plasma samples from 14 consecutive blacks with clinically quiescent chronic pancreatitis and 15 outwardly healthy hospital personnel at Soweto, the township near Johannesburg in South Africa. The patients had higher serum levels than did controls of lipid isomerisation (P < 0.002) and peroxidation (P < 0.05) markers, with lower plasma levels of glutathione (P < 0.0001) and bioactive fraction of vitamin C (P < 0.002). Lipid peroxide and non-bioavailable vitamin C concentrations in Sowetan patients were significantly higher than in their counterparts from Manchester, UK (P < 0.0001, P < 0.0005, respectively). These differences mirrored those in controls in that outwardly healthy Sowetans had much higher serum lipid peroxide levels than Manchester controls (P < 0.001) and much lower plasma concentration of vitamin C (P < 0.001) and hence of the bioavailable fraction ascorbate (P < 0.0002). Heightened free radical activity is thus a common denominator in chronic pancreatitis irrespective of geography, or putative aetiological factors whether alcoholism or idiopathic, since that ratio was approximately 95:5 at Johannesburg and 50:50 at Manchester. The further finding of subclinical oxidative stress in Sowetan controls and the endemic nature of chronic pancreatitis in that area supports the hypothesis that oxidative stress may be involved in its pathogenesis.