Strong and frequent T-cell responses to the minor allergen Phl p 12 in Spanish patients IgE-sensitized to Profilins.

BACKGROUND: Profilins are dominant pan-allergens known to cause cross-sensitization, leading to clinical symptoms such as pollen-food syndrome. This study aimed to determine the T-cell response to Phl p 12 in profilin-sensitized patients, by measuring the prevalence, strength and cross-reactivity to clinically relevant profilins. METHODS: The release of Phl p allergens from pollen was determined by mass spectrometry and immunochemistry. T-cell responses, epitope mapping and cross-reactivity to profilins (Phl p 12, Ole e 2, Bet v 2 and Mal d 4) were measured in vitro using PBMCs from 26 Spanish grass-allergic donors IgE-sensitized to profilin. Cross-reactivity was addressed in vivo using 2 different mouse strains (BALB/c and C3H). RESULTS: Phl p 12 and Phl p 1 are released from pollen simultaneously and in similar amounts. Both T-cell response frequency (17/26 donors) and strength were comparable between Phl p 12 and Phl p 1. T-cell cross-reactivity to other profilins correlated with overall sequence homology, and 2 immunodominant epitope regions of Phl p 12 were identified. Data from mice immunized with Phl p 12 showed that cross-reactivity to Bet v 2 was mediated by conserved epitopes and further influenced by additional genetic factors, likely to be MHC II. CONCLUSION: The strength, prevalence and cross-reactivity of T-cell responses towards Phl p 12 are comparable to the major allergen Phl p 1, which supports the hypothesis that T cells to Phl p 12 can play an important role in development of allergic symptoms, such as those associated with pollen-food syndrome.

The complexity of allergic patients' IgE repertoire correlates with serum concentration of allergen-specific IgE.

BACKGROUND: The governing factor of both effector-cell activation and facilitated antigen presentation is IgE-repertoire complexity (IgE-clonality, -affinity and -concentration). Yet, the compositions of individual IgE repertoires and correlation between IgE-repertoire complexity and establishment of allergic sensitization remain to be determined. OBJECTIVE: In complex formation assays with recombinant IgE, allergen and CD23(+) B cells, we assess the composition of serum IgE repertoires and examine the correlation between IgE-titre and IgE-repertoire complexity. METHODS: The capacity of sera, from house dust mite-sensitized individuals, to mediate IgE-Der p 2-CD23 complex formation on CD23(+) B cells was measured. In parallel experiments, the effect of supplementing each serum with one or more Der p 2-specific monoclonal recombinant IgE antibodies on complex formation was determined. RESULTS: Only sera with the highest concentration of Der p 2-specific IgE resulted in complex formation without supplementary recombinant IgE. Intermediately titred sera supported complex formation to various degrees when supplemented with individual recombinant IgE. The degree of complex formation depended on the affinity and epitope specificity of the recombinant IgE. Complex formation by combining serum and recombinant IgEs could not be obtained with sera of relatively low titres of specific IgE. However, these sera had the capacity to dramatically enhance the low complex formation achieved with pairs of affinity-engineered recombinant IgEs. CONCLUSION AND CLINICAL RELEVANCE: Serum IgE complexity can be indirectly assessed by combining sera with defined monoclonal IgEs in IgE-allergen-CD23 complex assays. The observed differences in epitope-coverage of Der p 2-specific serum-IgE in sera of different specific IgE titres indicate that increased IgE titres correlate with increased complexity of the IgE-repertoire. A detailed knowledge of the composition and complexity of allergen-specific IgE repertoires (and the relation to IgE titre), particularly in the early phase of sensitization, may be used to improve the prediction of the persistence and severity of allergic symptoms, as well as the progression of the Allergic March.

S100A8/A9 deficiency in nonhealing venous leg ulcers uncovered by multiplexed antibody microarray profiling.

BACKGROUND: Knowledge on the underlying mechanisms for nonhealing chronic wounds is fragmentary. OBJECTIVES: To increase our understanding of the pathogenesis, the relationship between healing ability and a large panel of proteins was studied using a specially designed wound-healing antibody-based microarray. METHODS: Wound fluid from nondiabetic patients with nonhealing venous leg ulcers was compared with that from patients with healing open granulating acute wounds. The high-throughput method enabled simultaneous measurement of the relative levels of 48 different proteins representing major categories of wound-healing modulators. RESULTS: Unexpectedly, several of the examined proteins, including various proinflammatory cytokines, proteinases and antiproteinases, were not significantly (P>0·001) changed in chronic wound fluid. For example, levels of matrix metalloproteinase-9 and one of its substrates type IV collagen were similar in the two groups. The wound fluid samples displayed similar degrees of fragmentation of fibronectin by Western blot analysis and the total fibronectin levels were doubled (P<0·001) in chronic compared with acute wounds. The increased fibronectin originated from α-smooth muscle actin-positive myofibroblasts and not from the circulation. S100A8/A9 was the sole protein that was reduced (P<0·001) in wound fluid from venous ulcers [median 226 µg mL(-1) (interquartile range 213-278)] compared with healing wounds [455 µg mL(-1) (382-504)], probably reflecting a difference in inflammatory cell composition. CONCLUSION: The molecular anomalies in chronic wounds are more subtle than the current paradigm and neither excessive proteinase activity nor deficiencies of examined extracellular matrix proteins, growth factors or angiogenic/angiostatic factors appear to contribute significantly to the nonhealing state of venous leg ulcers.

Immunohistochemical staining for thyroid peroxidase (TPO) of needle core biopsies in the diagnosis of scintigraphically cold thyroid nodules.

BACKGROUND: Cold thyroid nodules are common, in particular in iodine-deficient areas, but only a minority of them are malignant requiring surgery. Thyroid peroxidase (TPO) immunostaining of fine-needle aspiration cytology (FNAC) material has proven helpful in diagnosing cells from malignant lesions, but the procedure has its limitations in a routine setting. PURPOSE: To improve diagnosis and reduce surgery rate, the FNAC procedure was replaced by needle core biopsy (NCB), which was routinely stained for TPO by the monoclonal antibody mAb 47. MATERIALS AND METHODS: During a 5-year period 427 consecutive patients with a cold thyroid nodule were evaluated by ultrasound-guided NCB, which had been routinely stained for TPO in an automated immunostainer. Sensitivity and specificity and predictive values of the TPO immunostaining were estimated, based on the final diagnosis obtained from surgical resection. RESULTS: The majority of nodules with benign NCB diagnosis were not surgically removed, and thus a subgroup of 140 operated nodules formed the basis for the calculations. Sensitivity and specificity for benign and malignant lesions were 100% if the oxyphilic variant of adenomas and minimally invasive follicular carcinomas were excluded. By inclusion of these, the values fell to 89% and 97%, respectively. The predictive value of a positive test was 96% and the predictive value of a negative test was 97%. CONCLUSION: TPO immunostaining was found to be a valuable adjunct to morphology in the diagnosis of cold thyroid nodules of the nonoxyphilic type.

Reduced mortality for women with mammography-detected breast cancer in east Denmark and south Sweden.

The 5-year relative survival from breast cancer in Denmark is 10 percentage points lower than in Sweden. This difference has been demonstrated previously as being caused partly by more involved lymph nodes and larger tumours in Denmark. Sweden has had nationwide mammography-screening coverage since 1991, whereas this is still in its infancy in Denmark. In the search for an explanation for the remaining survival difference, patient delay was a likely candidate. This study compared patient delay and mammography-detection between two national regions. Data on patient delay and mammography were obtained from hospital records from 1989 and 1994, and analysed using Cox proportional hazard analysis of death within the first 5 years, with the factors age, country, delay/mammography detection and established patho-anatomic variables. A comparison of patient delay and mammography detection in 1989 and 1994 showed more mammography-detected tumours in south Sweden and more women with long delay in east Denmark. Mammography detection, but not long patient delay, had a significant effect on the death hazard when adjusting for patho-anatomic risk factors. The hazard ratio was not eliminated in 1989, but in 1994, the hazard ratio between east Denmark and south Sweden was reduced from 1.3 to 1.1. In conclusion, patient delay did not appear to have any effect on 5-year survival when adjusting for patho-anatomic factors, but tumour detection by mammography affected survival favourably and partly explained the survival difference between east Denmark and south Sweden.

Can the survival difference between breast cancer patients in Denmark and Sweden 1989 and 1994 be explained by patho-anatomical variables?--a population-based study.

Analyses of data from cancer registries have shown a 10% unit difference in 5-year relative survival between Danish and Swedish patients with breast cancer. This study investigates the effect of age and patho-anatomic variables on this survival difference. Hospital records were collected for women over 40 years of age diagnosed in 1989 or 1994 in east Denmark and south Sweden; patho-anatomical variables and survival were compared between 2289 Danish and 1715 Swedish women. Tumours were smaller, node-negative axillae more frequent and well-differentiated tumours almost 10% more frequent in Sweden. A superior 5-year relative survival in Sweden was found in the 50- to 79-year age group. The adjusted hazard rate ratio between countries was 1.7 in 1989 and 1.3 in 1994. Conditional survival after surviving the first 5 years was similar for the two countries. Adjusting for patho-anatomical variables reduced but did not eliminate the higher risk of death among the Danish patients. Higher population death rates could explain some but not all of the residual elevated risk for Danish women.

Dependence of morphology on agitation intensity in fed-batch cultures of Aspergillus oryzae and its implications for recombinant protein production.

We previously reported that, although agitation conditions strongly affected mycelial morphology, such changes did not lead to different levels of recombinant protein production in chemostat cultures of Aspergillus oryzae (Amanullah et al., 1999). To extend this finding to another set of operating conditions, fed-batch fermentations of A. oryzae were conducted at biomass concentrations up to 34 g dry cell weight/L and three agitation speeds (525, 675, and 825 rpm) to give specific power inputs between 1 and 5 kWm(-3). Gas blending was used to control the dissolved oxygen level at 50% of air saturation except at the lowest speed where it fell below 40% after 60-65 h. The effects of agitation intensity on growth, mycelial morphology, hyphal tip activity, and recombinant protein (amyloglucosidase) production in fed-batch cultures were investigated. In the batch phase of the fermentations, biomass concentration, and AMG secretion increased with increasing agitation intensity. If in a run, dissolved oxygen fell below approximately 40% because of inadequate oxygen transfer associated with enhanced viscosity, AMG production ceased. As with the chemostat cultures, even though mycelial morphology was significantly affected by changes in agitation intensity, enzyme titers (AGU/L) under conditions of substrate limited growth and controlled dissolved oxygen of >50% did not follow these changes. Although the measurement of active tips within mycelial clumps was not considered, a dependency of the specific AMG productivity (AGU/g biomass/h) on the percentage of extending tips was found, suggesting that protein secretion may be a bottle-neck in this strain during fed-batch fermentations.

[TPO immunostaining of the solitary, cold thyroid nodules]. / TPO-immunfarvning af den solitaere, kolde thyreoideaknude.

INTRODUCTION: The chance of malignancy in scintigraphically cold thyroid nodules is 2-24%. Differentiation between malignant and benign cytology is difficult. The aim of this study was to evaluate the ability of immunostaining (MoAB47--raised against thyroid peroxidase (TPO)) to differentiate between malignant and benign cells taken from cold thyroid nodules by fine needle aspiration biopsy (FNAB) in order to reduce the number of unnecessary thyroid operations. MATERIALS AND METHODS: One hundred and eighty-one patients (150 female) with a scintigraphically cold, solitary thyroid nodule were entered between 1993 and 1996. Fifty-seven were excluded for various reasons. Material removed by FNAB was stained with MoAB47 and routine staining. Staining of 80% or more of the cells was considered benign, less than 80% was considered malignant. Routine staining of operatively removed material was used as the final diagnosis. RESULTS: A pattern with negative TPO staining was found in all lesions that were subsequently proved to be malignant. In all but one, the lesions subsequently diagnosed as being benign stained positive for TPO. The sensitivity and specificity were respectively 1.0 and 0.99. CONCLUSION: TPO immunostaining of material removed by FNAB is a powerful tool in the differentiation between benign and malignant tumours.

[Untreated breast cancer in Denmark 1978-1995]. / Ubehandlet brystkraeft i Danmark 1978-1995.

INTRODUCTION: The lack of registration of women who have received no or alternative treatment for breast cancer has been criticised. No distinction is made in the Danish Cancer Register between these patients and those who only receive palliative treatment for other reasons, such as old age, advanced disease, and competing illnesses. We have estimated the number of women in this group of patients, who, in reality, had not received any treatment with the intent to cure under the health care system, and whether a meaningful analysis of survival for these patients is feasible. METHOD: All women with breast cancer diagnosed during the years 1978-1995 were extracted from the Cancer Register, and we isolated those who had been registered as having had no or only palliative treatment and who had survived for a minimum of 45 days after diagnosis. A search was made in the Danish Breast Cancer Co-operative Group register for unreported treatment and the residual group was followed up individually. RESULTS: Out of 49.058 women with histologically or cytologically verified breast cancer, the Cancer Register listed 840 women with no registered treatment of their disease. Of these, there were 103 cases of carcinoma in situ. A match with the DBCG register revealed that 188 women had nevertheless been operated on. Among the remaining 549 women, 99 were truly untreated, and for 77 of these the reasons given were another or advanced disease or old age. Only 22 women had initially declined treatment for no specific reason. Five of these had later decided on subsequent curative treatment, which leaves 17 women in the category "breast cancer untreated at her own request" ("untreated breast cancer at own will"). Nine are dead, five had their tumour excised at biopsy, and the remaining three are alive with tumours diagnosed by fine needle aspiration biopsy (1) or thru-cut biopsy (2) after 7.7 and 4 years, respectively. CONCLUSION: This report has shown that a survival analysis based on the Cancer Register of untreated breast cancer in relation to treated breast cancer is not meaningful. A true estimation of survival after untreated versus treated breast cancer can only be achieved through a randomised study, which would be unethical.

The class I metabotropic glutamate receptor antagonist, AIDA, improves short-term and impairs long-term memory in a spatial task for rats.

Effects of the class I selective metabotropic glutamate receptor antagonist, 1-aminoindan-1,5-dicarboxylic acid (AIDA), on spatial procedural learning and episodic short-term memory of rats were investigated in an appetitively reinforced 3-choice delayed match-to-position task. First, an acute intraperitoneal injection of AIDA (2 mg/kg) was given 20 min before a single training session of 20 trials using repeated reward position in one alcove out of three. AIDA caused facilitated short-term acquisition within such a session compared to saline treated controls. Secondly, injections were given before each of ten sessions (48 h intervals) also using constant reward position. The results showed AIDA induced inhibition of procedural between-session acquisition. Finally, the use of reward positions in a non-repetitive but trial-specific version of the 3-choice test revealed a facilitating effect of AIDA on episodic short-term memory.

Task-specific enhancement of short-term, but not long-term, memory by class I metabotropic glutamate receptor antagonist 1-aminoindan-1,5-dicarboxylic acid in rats.

Pharmacological application of broad agonists and antagonists has supported the notion of a potential role of metabotropic glutamate receptors (mGluRs) in learning and memory formation, but the specific function of the different classes or individual subtypes remains elusive. Furthermore, our knowledge with respect to different learning mechanisms is still fragmentary. In an attempt to clarify further the function of mGluRs in learning, rats were trained in various paradigms in the presence/absence of the specific class I antagonist 1-aminoindan-1,5-dicarboxylic acid (AIDA). Intraperitoneal application of AIDA prior to training led to enhanced within-session performance in animals trained in a positively reinforced reference memory task in a three-choice maze. However, this enhancement did not result in increased retention as measured by the number of correct responses during the first four trials of each session on subsequent days. The increase was purely an enhancement in within-session performance, required doses higher than 2 mg/kg, and was not accompanied by an unspecific increase in activity as monitored in the open field. By contrast, AIDA animals trained in a combined shock-reinforced contextual and cue conditioning paradigm demonstrated a pronounced retention deficit compared with controls in conditioning to the context, but not the cue (a high-frequency tone). Although within-session performance during context and cue periods was slightly increased in the AIDA group, the difference did not reach significance. Drug-induced hyperactivity, which could account for the memory deficit, was excluded by recordings of activity in specific activity cages. These results shed new light on the possible function of class I mGluRs in learning and memory formation and imply that systemic blockade of class I mGluRs may enhance short-term memory under certain learning conditions.

Semi-on-line analysis for fast and precise monitoring of bioreaction processes.

Monitoring of substrates and products during fermentation processes can be achieved either by on-line, in situ sensors or by semi-on-line analysis consisting of an automatic sampling step followed by an ex situ analysis of the retrieved sample. The potential risk of introducing time delays and signal bias during sampling makes it necessary to distinguish between real-time, on-line, in situ methods and semi-on-line analysis. In addition, semi-on-line analyzers are often mechanically complex--a circumstance which has to be given special attention during their industrial use on a routine basis. This review on semi-on-line analysis will focus both on the dynamics and precision of aseptic sampling devices and on the performance of flow injection analysis (FIA) and sequential injection analysis (SIA), especially with regard to their robustness when used in industry.

[Preoperative scintigraphic localization of hyperfunctioning parathyroid glands]. / Praeoperativ scintigrafisk lokalisation af hyperfungerende parathyreoideakirtler.

Preoperative identification of hyperfunctioning parathyroid glands was performed by 99m-Tc-sestamibi scintigrams in 29 patients with hyperparathyroidism. Out of 30 histopathologically proven diseased parathyroid glands 21 were identified by scintigraphy. The diagnostic specificity (PVpos) was 88%. All diseased glands weighing more than 1200 mg were identified by scintigraphy including four glands in the mediastinum. 99m-Tc-sestamibi scintigraphy can identify the larger hyperfunctioning parathyroid glands with high reliability. The method was of great value in situations with ectopic abnormal parathyroid glands.

[Mammographic screening in the municipality of Copenhagen. Results of the first screening round]. / Mammografiscreening i Københavns Kommune. Resultater fra første screeningsrunde.

The aim of the study was to evaluate the results from the prevalence round of the mammography screening programme in the Municipality of Copenhagen. All women who by 1 April 1991 were 50-69 years old, and who lived in the Municipality of Copenhagen, were during the period 1 April 1991-24 April 1993 offered a mammography. Those with suspect findings were recalled for further examination and possible biopsy. Women with breast cancer were offered treatment according to the standard national protocols (DBCCG). The participation rate was 71% (30,416/43,087). Among these 2043 (6.7%) were re-examined and 592 (1.9%) underwent surgical biopsy. Breast cancer was revealed in 359 (1.2%) women, of whom 88% had an invasive breast cancer. Prevalence of breast cancer increased significantly with increasing age. The positive predictive value for breast cancer among those re-examined was 18% and for those who had a surgical biopsy 61%. Among women with an invasive breast cancer 41% had a tumour of 10 mm or less, 80% had negative lymph node status and 56% had breast conserving surgery. During the following 12 months 14 women with a normal mammogram at the screening round developed breast cancer giving a sensitivity of 96%. It is concluded that the first mammography screening in Denmark showed the highest breast cancer prevalence published so far. A possible explanation could be a high sensitivity of the screening method, indicated by a relatively high frequency of small cancers. The screening programme was fully comparable with international standards.

Growth energetics and metabolic fluxes in continuous cultures of Penicillium chrysogenum.

Continuous cultures of the penicillin producing fungus Penicillium chrysogenum have been analyzed with respect to the macromolecular composition of the mycelium. All cultivations were carried out using a chemically defined medium with glucose as the growth limiting component. Biomass was harvested at steady state and analyzed for proteins, lipids, RNA, DNA, and carbohydrates. Carbohydrates present in the cell wall, i.e., glucans and chitin, and carbohydrates serving as storage materials, i.e., glycogen, were measured. It was observed that the levels of DNA and lipids are relative constant, whereas the proteins and stable RNA levels increase with the specific growth rate and the total amount of carbohydrates decreases with the specific growth rate. Glycogen is only present in small amounts, decreasing with the specific growth rate. As an average the measured macromolecules account for 77 +/- 2% (w/w) of the biomass. On the basis of estimations of the metabolic costs for biosynthesis and polymerization of the different macromolecules the total ATP and NADPH requirements for cell biosynthesis from glucose and inorganic salts, i.e., YxATP,growth and YxNADPH, have been quantified. The biosynthesis of 1 g biomass was calculated to require 39.9 mmol of ATP and 7.5 mmol of NADPH when cytosolic acetyl-CoA is formed from citrate by citrate lyase and oxaloacetate is recycled back into the TCA cycle. Other pathways of acetyl-CoA biosynthesis have been considered. The calculations show that the different biosynthetic routes for generating cytosolic acetyl-CoA have a significant influence on the theoretical value of ATP and NADPH requirements for cell biosynthesis. Combining a detailed stoichiometric model for growth and product formation of P. chrysogenum with experimental data on the macromolecular composition of P. chrysogenum and precise measurements of substrate uptake and product formation the intracellular flux distribution was calculated for different cultivation conditions.

Continuous cultivation of Penicillium chrysogenum. Growth on glucose and penicillin production.

A series of constant-mass, continuous cultivations of the penicillin producing mold Penicillium chrysogenum was carried out using a chemically defined medium with glucose as the growth-limiting component. The stoichiometry for growth of P. chrysogenum on glucose was characterized in terms of mass-yield and maintenance coefficients. Saturation kinetics with respect to glucose was used to describe the glucose consumption rate at steady-state conditions. Transient data indicate that the maximum rate of glucose consumption at a particular set of operating conditions is correlated to the metabolic 'capacity' of the mold as reflected by its intracellular RNA content. A progressive loss in the penicillin productivity in glucose limited chemostat cultures was correlated to the formation of two mutants. The two mutants were characterized by their sporulation when grown as surface cultures and by Southern dot-tests for delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS), isopenicillin-N synthase (IPNS) and acyl-CoA:6-APA acyltransferase (AT). The loss of penicillin productivity was caused by an increasing fraction of mutants which had lost the genes encoding for all three enzymes needed in the penicillin synthesizing pathway.

Degradation of pencillin-V in fermentation media.

In Industrial production of penicillin there is a noticeable loss of the product through degradation reactions. It is shown that the degradation of penicillin-V, both in a complex and in a chemically defined medium, can be separated into a phosphate-catalyzed conversion of penicillin-V to penicilloic-V acid, overlaid by at least one other reaction in which penicillin V is degraded to as yet unknown products. Parameter values for the phosphatecatalyzed degradation are found to be independent of the type of fermentation medium. The rate of formation of other degradation products of penicillin-V is found to be significantly higher in a complex fermentation medium with corn-steep liquor in a chemically defined medium. (c) 1994 John Wiley & Sons, Inc.

[Silicone breast implants]. / Silikonebrystproteser.

A brief review of the use of silicone breast implants, their structure, methods of implantation and complications is presented. Acute complications are rare, being mainly infection and hematoma. Long-term complications, on the contrary, are common, consisting mainly of capsular contracture around the prosthesis with subsequent pain and deformation of the breast. More rarely silicone granulomas form, and prosthesis rupture or herniation occurs. The importance of silicone leakage for these complications is discussed separately as well as the treatment of and prevention of capsular contracture and demonstration of silicone in tissue. A critical attitude towards the use of silicone breast implants, when these are used for purely cosmetic purposes, is recommended at present. New improved types of silicone breast implants are currently being tested clinically.