Twenty-two years of candidaemia surveillance: results from a Norwegian national study.

Several studies have reported an increased incidence of candidaemia and a redistribution of species, with a decrease in the number of Candida albicans isolates. In Norway, a prospective, national surveillance study of candidaemia has been ongoing since 1991. Data from the period 1991-2003 have been published previously. The aim of this study was to follow up the incidence, species distribution and antifungal susceptibility of Candida species isolates from blood cultures in the period 2004-2012, and compare them with the corresponding findings from the period 1991-2003. Blood culture isolates of Candida species from all medical microbiological laboratories in Norway were identified and susceptibility tested at the Norwegian Mycological Reference Laboratory. A total of 1724 isolates were recovered from 1653 patients in the period 2004-2012. Comparison of the two periods showed that the average incidence of candidaemia episodes per 100 000 inhabitants increased from 2.4 (1991-2003) to 3.9 (2004-2012). The increase in incidence in the latter period was significantly higher in patients aged >40 years (p 0.001), and a marked increase was observed in patients aged >60 years (p < 0.001). In conclusion, the average incidence in Norway over a period of 22 years modestly increased from 2.4 to 3.9 per 100,000 inhabitants, this being mainly accounted for by candidaemia in the elderly. The species distribution was stable, and the rate of acquired resistance was low.

The antimicrobial peptides lactoferricin B and magainin 2 cross over the bacterial cytoplasmic membrane and reside in the cytoplasm.

The localization of immunolabelled antimicrobial peptides was studied using transmission electron microscopy. Staphylococcus aureus and Escherichia coli were exposed to lactoferricin B (17-41), lactoferricin B (17-31) and D-lactoferricin B (17-31). E. coli was also exposed to cecropin P1 and magainin 2. The lactoferricins were found in the cytoplasm of both bacteria. In S. aureus the amount of cytoplasmic lactoferricin B (17-41) was time- and concentration-dependent, reaching a maximum within 30 min. Cecropin P1 was confined to the cell wall, while magainin 2 was found in the cytoplasm of E. coli. The finding of intracellularly localized magainin is not reported previously.

Post-antibiotic effect of the antimicrobial peptide lactoferricin on Escherichia coli and Staphylococcus aureus.

Lactoferricin is an antimicrobial peptide generated by gastric pepsin cleavage of lactoferrin. A possible post-antibiotic effect (PAE) of bovine lactoferricin (Lfcin B) and two shorter peptide derivatives against Staphylococcus aureus and Escherichia coli was studied. A drug removal technique involving centrifugation and washing was used. No PAE was found for Lfcin B against these two bacteria. The shorter derivatives had a short PAE against E. coli. They had a short negative PAE against S. aureus. In conclusion, the overall PAE is not overwhelming, but the small differences found between the different peptide-bacteria combinations could indicate that different peptide mechanisms of action might be present.

Lactoferricin B causes depolarization of the cytoplasmic membrane of Escherichia coli ATCC 25922 and fusion of negatively charged liposomes.

Antimicrobial peptides have been extensively studied in order to elucidate their mode of action. Most of these peptides have been shown to exert a bactericidal effect on the cytoplasmic membrane of bacteria. Lactoferricin is an antimicrobial peptide with a net positive charge and an amphipatic structure. In this study we examine the effect of bovine lactoferricin (lactoferricin B; Lfcin B) on bacterial membranes. We show that Lfcin B neither lyses bacteria, nor causes a major leakage from liposomes. Lfcin B depolarizes the membrane of susceptible bacteria, and induces fusion of negatively charged liposomes. Hence, Lfcin B may have additional targets responsible for the antibacterial effect.

Antibacterial effects of lactoferricin B.

The antimicrobial peptide, lactoferricin, can be generated upon gastric pepsin cleavage of lactoferrin. We have examined the inhibitory efficacy of lactoferricin of bovine origin (Lf-cin B) on Escherichia coli, Proteus mirabilis and Staphylococcus aureus with or without a cell wall. We found that spheroplasts and protoplasts had a lower MIC than their counterparts with a cell wall. We also compared the efficacies of Lf-cin B (17-31) made of all L-amino acids and all D-amino acids. The peptide made of all D-amino acids was more active than the corresponding L-enantiomer. Furthermore, we examined the influence of Lf-cin B on the motility of E. coli and the influence of temperature on the susceptibility of bacteria exposed to Lf-cin B. Bacteria exposed to sub-MIC of Lf-cin B lost their motility. Bacteria exposed to Lf-cin B at 20 degrees C were more sensitive to Lf-cin B than when exposed at 37 degrees C. These findings indicate that the cell envelope is a limiting step for Lf-cin B to exert its antibiotic effect. We cannot rule out a receptor-mediated first step for Lf-cin B (17-31).

Constant low rate of fungemia in norway, 1991 to 1996. The Norwegian Yeast Study Group.

Since 1991 information on yeast isolates from blood cultures has been recorded prospectively from all microbiological laboratories (5 university and 16 county or local hospital laboratories) in Norway (population, 4.3 million). From 1991 to 1996 a total of 571 episodes of fungemia in 552 patients occurred (1991, 109 episodes; 1992, 81 episodes; 1993, 93 episodes; 1994, 89 episodes; 1995, 98 episodes; and 1996, 101 episodes). The fungemia rates per 10,000 patient days were 0.29 in 1991 and 0.27 in 1996. The average rates for the years 1991 to 1996 were 0.37 for the university laboratories and 0.20 for the other laboratories. These rates are low compared to the rate (0. 76) in five Dutch university hospitals in 1995 and the rate (2.0) in Iowa in 1991. The four most frequently isolated species were Candida albicans (66%), Candida glabrata (12.5%), Candida parapsilosis (7.6%), and Candida tropicalis (6.4%). The incidences of both C. albicans (range, 63 to 73%) and C. glabrata (range, 8.4 to 15.7%) varied somewhat throughout this period, but no significant increase or decrease was noted. MICs of amphotericin B, flucytosine, and fluconazole were determined for 89% of the isolates. All were susceptible to amphotericin B, and only 29 (5.6%) strains had decreased susceptibility to flucytosine. All C. albicans isolates were susceptible to fluconazole. The percentage of yeast isolates with decreased susceptibility to fluconazole (MICs, >/=16 microgram/ml) did increase, from 9.6% in 1991 and 1992 to 12.2% in 1994, 16.1% in 1995, and 18.6% in 1996. This was largely due to increases in the percentages of resistant C. glabrata and Candida krusei strains in the last 2 years. Compared to the incidence in other countries, it is remarkable that Norway has such a low and constant incidence of fungemia. A possible reason for this difference might be a restricted antibiotic use policy in Norway.

Lactoferricin of bovine origin is more active than lactoferricins of human, murine and caprine origin.

The antimicrobial peptide lactoferricin is generated by gastric pepsin cleavage of lactoferrin. We have examined the antimicrobial activity of lactoferricins derived from lactoferrin of human, murine, caprine and bovine origin with minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) against E. coli ATCC 25922 and S. aureus ATCC 25923. We found that lactoferricin of bovine origin (Lf-cin B) was the most efficacious of the lactoferricins tested. By comparing the linear and cyclic Lf-cin B we found the cyclic peptide to be the most active. Lactoferricin B was moderately active against E. coli ATCC 25922 and S. aureus ATCC 25923, but had no activity against P. mirabilis or Y. enterocolitica. Lf-cin B showed good activity against C. albicans, C. tropicalis and C. neoformans.

Phosphoinositide metabolism in a polyoma-BK-virus-transformed pancreatic islet cell line: evidence for constitutively activated phospholipase C.

We have characterized the phosphoinositide metabolism in a polyoma-BK-virus-transformed rat pancreatic islet cell line which has highly malignant characteristics, expresses viral T-antigen and has lost insulin-secreting capacity. After incorporation with [3H]inositol to isotopic equilibrium, all inositol metabolites were analyzed. When compared with normal pancreatic islets, increased levels of inositol 1,4,5-trisphosphate (Ins-1,4,5-P3), inositol 1,3,4-trisphosphates and inositol tetrakisphosphate (Ins-P4), and decreased levels of phosphatidylinositol monophosphate (PIP) and phosphatidylinositol bisphosphate (PIP2) were found. The Ins-1,4,5-P3/PIP2 ratio increased, whereas the PIP2/PIP ratio was not altered after the transformation. In the pancreatic islet cell line there was a stable accumulation of inositol phosphates at 3.3 mM glucose. Glucose, KCl, cholecystokinin (CCK) and carbachol with and without LiCl were all without effect on the accumulation of inositol phosphates. Somatostatin inhibited the accumulation of inositol phosphates but a Ca(2+)-free/EDTA solution did not. Preincubation with cholera toxin or pertussis toxin inhibited the accumulation of inositol phosphates at 3.3 mM glucose except for Ins-P4, whereas no effect was observed on the phosphoinositides. NaF stimulated the accumulation of inositol phosphates, with a concomitant decrease in the phosphoinositides, whereas neomycin was without effect on the inositol phosphates. In normal pancreatic islets, pertussis toxin inhibited the CCK-induced increase in Ins-1,4,5-P3, whereas no effect was seen at 3.3 mM glucose. Finally, pertussis toxin inhibited the CCK-induced increase in the Ins-1,4,5-P3/PIP2 ratio in normal pancreatic islets. The same inhibition was also found in the pancreatic islet cell line at 3.3 mM glucose. We conclude that in the transformed pancreatic islet cell line the phosphoinositide hydrolysis is constitutively activated at the level of phospholipase C, with a substantial loss of regulatory control.

Transformed rat pancreatic islet-cell lines established by BK virus infection in vitro.

Single-cell suspensions of primary rat pancreatic islet cells were infected with BK virus (BKV) prototype (Gardner) and the naturally occurring BKV strain (TU), isolated from human urine. These viral strains have different sequences in their non-coding control regions which contain promoter-enhancer elements and origin of DNA replication. Uninfected cell cultures disintegrated within 3 weeks, while a varying number of virus-infected cultures were immortalized and a majority exhibited focal areas of 2-dimensional growth. A significantly higher proportion of the BKV (TU)- than of the BKV (Gardner)-infected cultures were immortalized. Large tumor (T) antigen expression was evident in virus-infected cultures from day 4 post infection (p.i.), while insulin secretion decreased steadily during the first weeks of culture. Two cell lines were established from BKV (TU)-infected cultures. Line 5A4 was contact-inhibited, growing as a dense monolayer, while 6A3 demonstrated foci of 2-dimensional growth. Both cell lines have retained their morphology and T-antigen expression for approximately 130 passages. At high passages a low level of intracellular insulin, but no secretion into media, was detected. Based on standard biological tests (reduced serum requirements, growth at low cell density, anchorage-independent growth and tumor induction in nude mice) both cell lines have a fully transformed phenotype, but 6A3 appears to be more malignantly transformed. Since both cell lines were established simultaneously from the same primary cells with the same virus batch, they provide an opportunity to study the transforming mechanisms of BKV in a relative context.

Changes in postprandial release patterns of gastrointestinal hormones in late pregnancy and the early postpartum period.

Intestinal transit time increases and gastrointestinal incretin effect is reported to decrease in pregnancy. The release patterns of gastrointestinal hormones related to these functions were studied in eight women before and after ingestion of a standardized meal at 32-34 weeks gestation and at 4 days postpartum. Basal plasma motilin and the integrated meal response of motilin, pancreatic polypeptide (PP) and gastric inhibitory polypeptide (GIP) were significantly lower in pregnancy than postpartum. The meal-induced rise of somatostatin and vasoactive intestinal polypeptide (VIP) was, however, absent in late pregnancy; whereas the somatostatin response recovered postpartum, and the plasma VIP concentrations stabilized at significantly higher levels postpartum without any meal response. Basal and meal-induced plasma insulin were significantly higher in pregnancy.

Brain-gut peptides in sauna-induced hyperthermia.

The release of brain-gut peptides during sauna bathing was studied in seven women. All women underwent a 20 min sauna bath. Their sublingual temperature rose from 36.9 +/- 0.1 degrees C to 38.6 +/- 0.2 degrees C (mean +/- SEM). A significant increase in circulating plasma vasoactive intestinal polypeptide (VIP) was observed during heat exposure, whereas plasma pancreatic polypeptide (PP), motilin and blood glucose rose and stayed significantly elevated first during the ensuing 60 min (P less than 0.05 in all cases). A similar increase in plasma insulin failed to reach statistical significance, whereas the plasma levels of somatostatin and cholecystokinin (CCK) remained unchanged. It is suggested that the plasma VIP levels are related to compensatory mechanisms during heat exposure with vasodilatation and heat loss.

Characterization of secretin-like immunoreactivity in pregnancy.

The plasma secretin-like immunoreactivity (SLI) in 23 healthy females was elevated in late pregnancy (34 +/- 3 pmol/l) as compared with 23 non-pregnant female controls (12 +/- 2 pmol/l; p less than 0.01). The plasma SLI in pregnancy eluted close to albumin on a Sephadex G-200 column, whereas 50-75% of the recovered SLI was displaced to the elution volume of free secretin when plasma was exposed to 6 mol/l urea. When 125I-labelled secretin was incubated with plasma in the absence of secretin antibodies, 40% of the intact label eluted in the void volume of a Sephadex G-50 Fine column in pregnancy, compared with only 18% in the nonpregnant state. The present study supports the notion that secretin circulates bound to plasma proteins and suggests that the protein binding of secretin is enhanced in late pregnancy, a feature common to several classical hormones.

The effect of severe pre-eclampsia on the pancreas: changes in the serum cationic trypsinogen and pancreatic amylase.

In 13 patients with severe pre-eclampsia mean serum concentrations of cationic trypsinogen and amylase were statistically significantly higher at 64 ng/ml and 1.6 mumol/l, respectively, than the corresponding mean serum concentrations in 30 normal pregnancies, 22 ng/ml and 1.1 mumol/l respectively. These rises exceeded the expected increased values due to the slight reduction of the renal function in the pre-eclamptic patients, and therefore indicate a concomitant injury of the pancreas.

Evidence of somatostatin as a humoral modulator of motilin release in man. A study of plasma motilin and somatostatin during intravenous infusion of somatostatin, secretin, cholecystokinin, and gastric inhibitory polypeptide.

In six healthy persons receiving graded intravenous infusions of synthetic somatostatin the plasma motilin concentrations decreased significantly (p less than 0.05) already with doses giving physiological plasma somatostatin levels, and a rebound of plasma motilin was observed after cessation of infusion of pharmacological somatostatin doses. After an intravenous secretin infusion (280 pmol/kg-h) producing pharmacological plasma secretin concentrations, a comparable plasma somatostatin increase was observed together with a substantial decrease in plasma motilin (p less than 0.05). Infusion of cholecystokinin in a pharmacological dose and of gastric inhibitory polypeptide (GIP) in doses giving plasma GIP levels in the physiological range had no effect on plasma somatostatin or motilin. Circulating plasma somatostatin may be a physiological modulator of the motilin release, and the plasma motilin fall seen during infusion of pharmacological doses of secretin may possibly be explained by the secretin-induced somatostatin release occurring simultaneously.

Effect of proximal gastric vagotomy on insulin- induced gastric H+ and pepsin secretion and serum group I pepsinogens.

The effect of insulin-induced hypoglycemia on serum group I pepsinogens (PG I), gastric H+, and pepsin secretion was studied in 11 duodenal ulcer patients before and 30 days after a proximal gastric vagotomy. The hypoglycemia elicited a significant increase in all the three variables preoperatively but in none of them postoperatively. This suggests that the preoperative increase in serum PG I must be mediated by the vagal nerves. Four of the 11 patients had an incomplete vagotomy by the method of Hollander. Serum PG I did not show any rising tendency in these patients postoperatively. It seems that serum PG I is a less sensitive indicator of the "completeness" of vagotomy than gastric H+ output. The serum PG I response to insulin-induced hypoglycemia can accordingly not be used as a clinical test for completeness of vagotomy.

Carcinoma diagnosed in excised gastric ulcers.

Seven patients with ulcer cancer are reported. Three were peroperatively, one of them also preoperatively, suspected of malignancy and showed carcinomatous infiltration of the gastric wall, penetrating deep into the subserous layer in all cases, and lymph node infiltration in two. These patients died of their cancer, respectively 1/2, 2 and 41/2 years after operation. Four patients showed peroperative gross signs of benign gastric ulcer. In 3 of the cases carcinoma, respecting the subserous layer, was found histologically in the excised ulcer. After subsequent radical surgery in 2 and biopsy from the site of ulcer excision in 1, carcinoma was not found in the specimens. In the fourth case an excised piece of a prepyloric ulcer on the posterior wall showed superficial carcinoma. After subsequent radical operation persistent carcinoma was found in the resected specimen at the site of the biopsy. The 4 patients are alive without signs of cancer, respectively 31/2, 5, 8 and 8 years after the operation. The series demonstrates that histological examination of excised gastric ulcers may lead to the diagnosis of a carcinoma, which has limited extension and good prognosis after operative treatment.