In vivo characterization of pustules in Malassezia Folliculitis by reflectance confocal microscopy and optical coherence tomography. A case series study.

BACKGROUND AND OBJECTIVE: Malassezia Folliculitis (MaF) is an inflammatory condition of hair follicles caused by Malassezia yeast. Optical coherence tomography (OCT) and reflectance confocal microscopy (RCM) are imaging technologies enabling in vivo visualization of superficial skin layers. This study explores morphology of pustules in MaF imaged by OCT and RCM. METHODS: Patients with microscopically verified MaF were included in this case series. Morphology was evaluated qualitatively with RCM and OCT, focusing on shape, border and content of selected pustules. RESULTS: Nine patients with MaF were included. Clinically, six patients presented monomorphic MaF with multiple superficial pustules, while three patients showed more polymorph MaF appearance. In total 13 pustules were investigated by RCM and OCT. In RCM images, pustules varied from having a well-defined border with homogenous content to ill-defined borders with heterogeneous content. A distinct black halo was occasionally observed around pustules as were dilated vessels. In OCT images, pustules appeared polymorphic, showing both well- and ill-defined structures with oval or irregular shape and more or less homogenous content. Malassezia fungi were not discernible by either RCM or OCT. Specific morphological image features in RCM and OCT did not reflect different clinical manifestations of MaF. CONCLUSION: RCM and OCT images identify morphological aspects of MaF pustules, and confirm that MaF is a folliculitis with clinical as well as morphological variance.

Optimum bolus wizard settings in insulin pumps in children with Type 1 diabetes.

AIM: To evaluate current insulin pump settings in an optimally regulated paediatric population using bolus wizard. METHODS: We used a retrospective study design to analyse data from 124 children on insulin pump therapy who had optimum HbA1c levels [< 59 mmol/mol (< 7.5%)] and no history of severe hypoglycaemic events. Bolus wizard settings were used to calculate the insulin to carbohydrate factors and insulin sensitivity factors. Multiple regression analysis was used to analyse the variables associated with the calculation factors. RESULTS: Insulin to carbohydrate factor varied from 276 in the youngest group to 424 in the oldest group, and increased according to age. Insulin sensitivity factor was highest in the group aged 6 to < 12 years, with a value of 125. Age, amount of carbohydrates, number of boluses per day and insulin per kg were all significantly associated with both calculation factors. Furthermore, duration of insulin pump treatment was significantly associated with insulin sensitivity factor and percentage bolus/basal was significantly associated with insulin to carbohydrate factor. Gender, diabetes duration and BMI were not associated with any of the calculation factors. CONCLUSION: Optimum insulin pump settings at pump initiation depend on both insulin requirements and use of the pump. Settings need to be individualized because the standardized calculation factors are not constant for children. There is a need to develop specific age- and insulin dose-dependent calculation factors.

Complement activation cascade triggered by PEG-PL engineered nanomedicines and carbon nanotubes: the challenges ahead.

Since their introduction, poly(ethylene glycol)-phospholipid (PEG-PL) conjugates have found many applications in design and engineering of nanosized delivery systems for controlled delivery of pharmaceuticals especially to non-macrophage targets. However, there are reports of idiosyncratic reactions to certain PEG-PL engineered nanomedicines in both experimental animals and man. These reactions are classified as pseudoallergy and may be associated with cardiopulmonary disturbance and other related symptoms of anaphylaxis. Recent studies suggest that complement activation may be a contributing, but not a rate limiting factor, in eliciting hypersensitivity reactions to such nanomedicines in sensitive individuals. This is rather surprising since PEGylated structures are generally assumed to suppress protein adsorption and blood opsonization events including complement. Here, we examine the molecular basis of complement activation by PEG-PL engineered nanomedicines and carbon nanotubes and discuss the challenges ahead.

Identification of Fmoc-beta-Ala-OH and Fmoc-beta-Ala-amino acid-OH as new impurities in Fmoc-protected amino acid derivatives.

During the manufacture of a proprietary peptide drug substance a new impurity appeared unexpectedly. Investigation of its chemical structure established the impurity as a beta-Ala insertion mutant of the mother peptide. The source of the beta-Ala was identified as contamination of the Fmoc-Ala-OH raw material with Fmoc-beta-Ala-Ala-OH. Further studies also demonstrated the presence of beta-Ala in other Fmoc-amino acids, particularly in Fmoc-Arg(Pbf)-OH. In this case, it was due to the presence of both Fmoc-beta-Ala-OH and Fmoc-beta-Ala-Arg(Pbf)-OH. It is concluded that beta-Ala contamination of Fmoc-amino acid derivatives is a general and hitherto unrecognized problem to suppliers of Fmoc-amino acid derivatives. The beta-Ala is often present as Fmoc-beta-Ala-OH and/or as a dipeptide, Fmoc-beta-Ala-amino acid-OH. In collaboration with the suppliers, new specifications were introduced, recognizing the presence of beta-Ala-related impurities in the raw materials and limiting them to acceptable levels. The implementation of these measures has essentially eliminated beta-Ala contamination as a problem in the manufacture of the drug substance.

Optimization of preparation of mitochondria from 25-100 mg skeletal muscle.

A method for isolation of mitochondria from 25-100 mg skeletal muscle is described. The instrumental developments include a refined homogenization setup, special handling techniques, and equipment for biopsy storage. The preparation medium was a standard ionic medium. All fractions were assayed for marker enzymes and the data used in optimization of the yield. It was observed that the homogenization procedure exerts strong control on the integrity of the isolated mitochondria. The method was developed with pigeon breast muscle as the model tissue and used virtually unaltered for preparation from muscles of pigs, rats, and humans. The relative yield was 40-50% and the mitochondria were well coupled and showed high rates of phosphorylating respiration.

Characterization of mitochondria from pig muscle: higher activity of exo-NADH oxidase in animals suffering from malignant hyperthermia.

Mitochondria were isolated from biopsies of the biceps femoris muscle of Danish landrace pigs. Three groups of animals were compared: (1) normal pigs; (2) pigs that were homozygous with respect to the gene Hal(n)/Hal(n) coding for the porcine malignant hyperthermia syndrome; and (3) heterozygote animals. A newly developed micro-method for preparation and assaying of small quantities of intact mitochondria was employed. With this technique mitochondria from biopsies weighing less than 100 mg were examined with respect to cytochrome content as well as phosphorylating and respiratory activities, including the nonphosphorylating exo-NADH oxidase activity. The mitochondria, prepared in a yield of 48%, showed high respiratory activities with tricarboxylic acid-cycle intermediates and pyruvate, and somewhat lower activity with palmitoyl-carnitine as substrate. The ATP synthase activity was about 1000 micromol ATP/min per g of protein and the maximal respiratory activity approx. 700 micromol of O2/min per g of protein. No differences among the three groups of animals were detected, except for the exo-NADH oxidase activities, which were 43, 78 and 107 micromol of O2/min per g of protein in the groups of normal, heterozygous and homozygous animals respectively. It is concluded that the exo-NADH oxidase activity may be a genetic manifestation of malignant hyperthermia and may play a significant role in the heat production characteristic of the syndrome.

Flow cytometric DNA analysis of breast cancers with predominance of carcinoma in situ: a comparison of the premalignant and malignant components.

Flow cytometric DNA analysis was performed on unfixed frozen tissue samples from 48 cases of invasive breast cancer (IC) with a predominance of ductal carcinoma in situ (DCIS). In 15 cases the samples contained only the DCIS component, in 17 cases only the IC component, whereas in 16 cases separate samples from the DCIS as well as the IC part within the individual lesion were available. In the latter 16 cases, complete or partial accordance in DNA ploidy between DCIS and IC was found in 12 cases, whereas no correspondence could be demonstrated in the remaining 4 cases, possibly due to intratumoral DNA heterogeneity. Comparison of the DNA index distribution in samples of DCIS and IC from the 48 cases showed concordant results except for the DNA hyperdiploid subclass, in which 6 clones were found in the DCIS portion compared to 18 clones in the IC portion. S-phase fractions were also comparable in the two groups. A comparison of the DCIS component from the present series of breast cancers to our previous series of pure DCIS also showed similar results with respect to the DNA index distribution, DNA heterogeneity, and S-phase fraction. No differences could be demonstrated between DCIS with and without invasion. The results indicate that the DNA ploidy pattern of breast cancer, as detected by flow cytometric DNA analysis, is established at the preinvasive stage of carcinogenesis.

[Blood transfusion increases the risk of surgical infection]. / Blodtransfusion øger den kirurgiske infektionsrisiko.

In a prospective randomized trial the frequency of infectious complications and natural killer cell function were investigated in 197 patients undergoing elective colorectal surgery and having either no blood transfusion (n = 93), transfusion with whole blood (n = 56), or filtered blood free from leucocytes (n = 48). Postoperative infections developed in 13 patients transfused with whole blood (23%), in one patient transfused with blood free from leucocytes (2%) and in two non-transfused patients (2%) (p < 0.01). Natural killer cell function was significantly (p < 0.001) impaired up to 30 days after surgery in patients transfused with whole blood. These data provide a strong case against the use of whole blood transfusion in patients undergoing elective colorectal surgery.

Postoperative infection and natural killer cell function following blood transfusion in patients undergoing elective colorectal surgery.

The frequency of infection in 197 patients undergoing elective colorectal surgery and having either no blood transfusion, transfusion with whole blood, or filtered blood free from leucocytes and platelets was investigated in a prospective randomized trial. Natural killer cell function was measured before operation and 3, 7 and 30 days after surgery in 60 consecutive patients. Of the patients 104 required blood transfusion; 48 received filtered blood and 56 underwent whole blood transfusion. Postoperative infections developed in 13 patients transfused with whole blood (23 per cent, 95 per cent confidence interval 13-32 per cent), in one patient transfused with blood free from leucocytes and platelets (2 per cent, 95 per cent confidence interval 0.05-11 per cent) and in two non-transfused patients (2 per cent, 95 per cent confidence interval 0.3-8 per cent) (P less than 0.01). Natural killer cell function was significantly (P less than 0.001) impaired up to 30 days after surgery in patients transfused with whole blood. These data provide a strong case against the use of whole blood transfusion in patients undergoing elective colorectal surgery.

Measurement of anal cross-sectional area and pressure during anal distension in healthy volunteers.

A probe for simultaneous measurement of cross-sectional area and pressure was used to elucidate biomechanical wall properties during anal distension. Measurements in distal sphincter regions demonstrated a high resistance to stretch and a large hysteresis compared to proximal sphincter regions. Resistance to stretch decreased during anal distension indicating an active relaxation mechanism at all recording levels. The zone with high resistance to stretch was located more distal than the high-pressure zone measured by anal-pressure profilometry. In conclusion, biomechanical wall properties of the anal canal cannot be described by classic viscoelastic theories but rather by a loss of sphincter tone caused by reflex mechanism during anal distension.

Vesico-ureteral reflux. I. Renal function and morphology in relation to anamnestic and urodynamic parameters.

Hundred and one children aged 12 years or less were consecutively referred with non-neurogenic vesico-ureteral reflux. None of the patients were previously operated for reflux. Based on the degree of reflux and the degree of nephropathy judged from urograms or renoscintigrams the patients were assigned an index defined as the sum status of both nephro-ureteric units. We found no correlation between VUR index, nephropathy index and GFR on one side, and bladder function parameters (spontaneous voiding, cystometry, bladder activity during sleep) on the other. Further, no correlation was found to history concerning urinary tract infections or enuresis the year before admittance. Possible reasons for the lack of correlation are discussed.

Vesico-ureteral reflux. II. The longterm outcome of kidney function in non-surgical treatment.

Seventy one patients were included in a prospective study of non-surgical treatment of vesico-ureteral reflux (VUR). We found 111 refluxing ureters, with 20 grade 1, 4 grade 2, 41 grade 3, 5 grade 4 and 1 grade 5. There was no correlation between presenting symptoms and degree of VUR or nephropathy, or between renal function in terms of glomerular filtration rate. During followup the patients were treated with long-term low-dose antibiotic prophylaxis and voiding disorders were corrected pharmacologically or by micturitional training. With a median follow up time of 6 years (3-10) 63% of the patients had cessation or down grading of VUR, irrespective of initial VUR degree. There was no correlation between cessation rate and urodynamic parameters, GFR, history of enuresis or previous urinary infections. The study supports a non-surgical attitude towards VUR patients irrespective of VUR degree, with prophylaxis of urinary infections and possibly correction of voiding disorders.

Computer analysis of manometric recordings. A study of overnight rectal activity in normal children.

To read and analyze manometric tracings from selected biological systems, a computer program was produced using low cost personal computer hardware. The program contains facilities for automatic scoring of contractions, intercontractile intervals (modality) and frequency analysis. This report concerns analysis of two overnight rectal motility recordings in 11 normal teenagers. After A/D conversion of data the developed software offers a set of options i.e. automatized scoring of contraction, detection of intervals between contractions and analytical tools such as histographic presentation of data, and presentation in the time domain. The A/D conversion time and computer analysis time for an 8 hour recording was 15 and 13 minutes, respectively. The analysis of frequency distribution overnight showed for the material as a whole 63% of no activity, 33% of 0-2 contractions x min-1 and 4% of more than 2 contractions x min-1. Modality analysis showed a peak between 15 and 25 seconds. Approximately 30% of the tracings showed accumulated intervals, which were multiples of the peak. No significant correlation was found between the first and second night of observation regarding the number of contractions and maximum frequency overnight. Significant correlation was found for the number of periods with high frequency activity between the two nights (p less than 0.05). Long-term pressure activity can be analyzed using the presented computer-aided method, providing a rapid and objective measurement of classical parameters and access to more in-depth analysis. This actual study of normal overnight rectal activity showed a large inter- and intra-individual variation.