Clinical relevance of neurological evaluation in patients suffering urinary retention in the absence of subvesical obstruction.

PURPOSE: Clinical relevance of neurological evaluation in patients suffered urinary retention in the absence of subvesical obstruction. Determining whether (1) women complaining residual bladder volume without prolapse and obstruction always suffer pudendal nerve damage; (2) neurogenic damage can be linked to patients history/clinical examination; (3) therapy alters regarding to neurological findings; and (4) electromyography (EMG) of musculus sphincter ani externus (MSAE) can be omitted with electronically stimulated pudendal nerve latency (ESPL) as the standard investigation. METHODS: Women with urinary retention without ≥stage 2 prolapse or obstruction have neurological investigation including vaginally and anally pudendal terminal nerve latency (PTNL) (>2.4 ms considered abnormal) and EMG seen 7/2005-04/2010. RESULTS: (1) 148/180 (82.2%) suffered at least moderate neurogenic damage and (2) severe neurogenic damage occurs with urge odds ratio (OR) = 3.1 or age (OR = 3.2). Correlations: spasticity with therapy changes (OR = 11.1), latencies. (a) Anally: (i) right and peripheral neuropathy (PNP) (OR = 2.5), chemotherapy (OR = 5.0); (ii) left and PNP (OR = 3.9), chemotherapy (OR = 4.8); (iii) left or right with PNP (OR = 3.9), chemotherapy (OR = 6.8); and (iv) left and right with chemotherapy (OR = 5.0). (b) Vaginally: (i) right with age >60 (OR = 3.2), radical operation (OR = 10.6); (ii) left with diabetes mellitus (OR = 2.5); and (iii) left or right with age (OR = 3.3), radical operation (OR = 8.7). (3) 19.6% therapy changes (36 patients). (4) Neither EMG nor ESPL can be replaced one by another (p = 0.12 anal, p = 0.05 vaginal). CONCLUSION: Red flags are neurogenic damage, age >60, chemotherapy, PNP, radical operation or diabetes. In unclear situations, EMG and ESPL need to be performed to gain relevant information.

GA(2)LEN skin test study I: GA(2)LEN harmonization of skin prick testing: novel sensitization patterns for inhalant allergens in Europe.

BACKGROUND: Skin prick testing is the standard for diagnosing IgE-mediated allergies. However, different allergen extracts and different testing procedures have been applied by European allergy centres. Thus, it has been difficult to compare results from different centres or studies across Europe. It was, therefore, crucial to standardize and harmonize procedures in allergy diagnosis and treatment within Europe. AIMS: The Global Asthma and Allergy European Network (GA(2)LEN), with partners and collaborating centres across Europe, was in a unique position to take on this task. The current study is the first approach to implement a standardized procedure for skin prick testing in allergies against inhalant allergens with a standardized pan-European allergen panel. METHODS: The study population consisted of patients who were referred to one of the 17 participating centres in 14 European countries (n = 3034, median age = 33 years). Skin prick testing and evaluation was performed with the same 18 allergens in a standardized procedure across all centres. RESULTS: The study clearly shows that many allergens previously regarded as untypical for some regions in Europe have been underestimated. This could partly be related to changes in mobility of patients, vegetation or climate in Europe. CONCLUSION: The results of this large pan-European study demonstrate for the first time sensitization patterns for different inhalant allergens in patients across Europe. The standardized skin prick test with the standardized allergen battery should be recommended for clinical use and research. Further EU-wide monitoring of sensitization patterns is urgently needed.

GA(2)LEN skin test study II: clinical relevance of inhalant allergen sensitizations in Europe.

BACKGROUND: Skin prick testing is the standard for diagnosing IgE-mediated allergies. A positive skin prick reaction, however, does not always correlate with clinical symptoms. A large database from a Global Asthma and Allergy European Network (GA(2)LEN) study with data on clinical relevance was used to determine the clinical relevance of sensitizations against the 18 most frequent inhalant allergens in Europe. The study population consisted of patients referred to one of the 17 allergy centres in 14 European countries (n = 3034, median age = 33 years). The aim of the study was to assess the clinical relevance of positive skin prick test reactions against inhalant allergens considering the predominating type of symptoms in a pan-European population of patients presenting with suspected allergic disease. METHODS: Clinical relevance of skin prick tests was recorded with regard to patient history and optional additional tests. A putative correlation between sensitization and allergic disease was assessed using logistic regression analysis. RESULTS: While an overall rate of >or=60% clinically relevant sensitizations was observed in all countries, a differential distribution of clinically relevant sensitizations was demonstrated depending on type of allergen and country where the prick test was performed. Furthermore, a significant correlation between the presence of allergic disease and the number of sensitizations was demonstrated. CONCLUSION: This study strongly emphasizes the importance of evaluating the clinical relevance of positive skin prick tests and calls for further studies, which may, ultimately, help increase the positive predictive value of allergy testing.

GA2LEN skin test study III: minimum battery of test inhalent allergens needed in epidemiological studies in patients.

BACKGROUND: The number of allergens to be tested in order to identify sensitized patients is important in order to have the most cost-effective approach in epidemiological studies. OBJECTIVE: To define the minimal number and the type of skin prick test (SPT) allergens required to identify a patient as sensitized using results of the new Pan-European GA(2)LEN skin prick test study. METHOD: In a large Pan-European multicenter (17 centers in 14 countries) patient based study, a standardized panel of 18 allergens has been prick tested using a standardized procedure. Conditional approach allowed to determine the allergens selection. RESULT: Among the 3034 patients involved, 1996 (68.2%) were sensitized to at least one allergen. Overall, eight allergens (grass pollen, Dermatophagoides pteronyssinus, birch pollen, cat dander, Artemisia, olive pollen, Blatella and Alternaria) allowed to identified more than 95% of sensitized subjects. However, differences were observed between countries, two allergens being sufficient for Switzerland (grass pollen and cat dander) as opposed to nine for France (grass pollen, Dermatophagoides pteronyssinus, olive pollen, cat dander, Blatella, cypress, dog dander, alder and [Artemisia or Alternaria]). According to country, up to 13 allergens were needed to identify all sensitized subjects. CONCLUSION: Eight to ten allergens allowed the identification of the majority of sensitized subjects. For clinical care of individual patients, the whole battery of 18 allergens is needed to appropriately assess sensitization across Europe.

Food-induced anaphylaxis and cofactors - data from the anaphylaxis registry.

Food allergens are frequent causes of anaphylaxis. In particular in children and adolescents they are the most frequent elicitors of severe allergic reactions, and in adults food allergens rank third behind insect venom and drugs. Since July 2006 severe allergic reactions from Germany, Austria, and Switzerland are collected in the anaphylaxis registry. Currently 78 hospitals and private practises are connected. From July 2006 until February 2009 1,156 severe allergic reactions were registered. Among children and adolescents (n = 187, age range from 3 months to 17 years) food allergens were the most frequent triggers, comprising 58% of cases. In the adult group (n = 968, 18 - 85 years) food allergens were in the third position (16.3%) behind insect venom and drugs. In children legumes (31%) and in particular peanuts were frequently responsible food allergens, followed by tree nuts (25%) with hazelnut being the most frequent elicitor. In adults fruits (13.4%) most often induced severe food-dependent anaphylaxis, but also animal products (12.2%); among these most frequently crustaceans and molluscs. Cofactors were often suspected in food-dependent anaphylaxis, namely in 39% of the adult group and in 14% of the pediatric group. In adults drugs (22%) and physical activity (10%) were reported to be the most frequent cofactors, in children physical activity was suspected in 8.7% and drugs in 2.6%. Concomitant diseases like atopic dermatitis, allergic asthma, or allergic rhinoconjunctivitis were reported in 78% of children and adolescents and in 67% of the adults. In conclusion, food-induced anaphylaxis, its cofactors and concomitant diseases are age-dependent. The data offers to identify risk factors of anaphylaxis.

Insect immune activation by recombinant Galleria mellonella apolipophorin III(1).

Apolipophorin III (apoLp-III) is an exchangeable insect apolipoprotein. Its function, as currently understood, lies in the stabilization of low-density lipophorin particles (LDLp) crossing the hemocoel in phases of high energy consumption to deliver lipids from the fat body to the flight muscle cells. Recent studies with native Galleria mellonella-apoLp-III gave first indications of an unexpected role of that protein in insect immune activation. Here we report the immune activation by the recombinant protein, documenting a newly discovered correlation between lipid physiology and immune defense in insects. The complete cDNA sequence of G. mellonella-apoLp-III was identified by mixed oligonucleotide-primed amplification of cDNA (MOPAC), 3'-RACE-PCR, and cRACE-PCR. The sequence coding for the native protein was ligated into a pET-vector; this construct was transfected into Escherichia coli and overexpressed in the bacteria. Photometric turbidity assays with human low density lipoprotein (LDL) and transmission electron microscopy studies on apoLp-III-stabilized lipid discs revealed the full functionality of the isolated recombinant apoLp-III with regard to its lipid-association ability. For proving its immune-stimulating capacity, apoLp-III was injected into the hemocoel of last instar G. mellonella larvae and the antibacterial activity in cell-free hemolymph was determined 24 h later. As a result, the hemolymph samples of injected insects contained strongly increased antibacterial activities against E. coli as well as clearly enhanced lysozyme-like activities. From Northern blot analysis of total RNA from insects injected with apoLp-III or the bacterial immune provocator lipopolysaccharide, it could be concluded that the transcription rate of apoLp-III mRNA does not vary in comparison to untreated last instar larvae.

A fast and reliable detection of indigo in historic and prehistoric textile samples.

Across all cultures and over all time periods, as early as the third millennium BC, indigo, a very intense blue vat dye, has been used to dye textiles. Even today, indigo is regarded as one of the most popular blue colorants in items such as blue jeans. While synthetic indigo is used to color textiles today, throughout history, a variety of plants have provided indigo. In this special feature article, René Csuk and co-authors propose to use atmospheric solids analysis probe mass spectrometry (ASAP-MS) to very rapidly and reliably identify indigo colorants tiny amounts in ancient historic fabrics without any sample preparation. The ionization in ASAP experiments is effected by Penning ionization or by the transfer of protons originating from protonated clusters of water. Dr. René Csuk is Professor of organic bioorganic chemistry at the Martin-Luther Universität Halle-Wittenberg (Halle, Germany). His main research interests are in organic and medicinal chemistry, chemical biology and phytochemistry. Graphical Abstract: Historical samples can be investigated conveniently using ASAP®-MS experiments. ASAP®-MS allowed for rapid direct sampling without any preparation of the sample prior to its measurements, and reliable results were generated in less than 1 minute. As demonstrated for indigoid-type dyes, the efficacy of ASAP®-MS analysis is independent of the type of fiber, the age of the sample, the workmanship, and the state of preservation.

Induction of immunity by latex beads and by hemolymph transfer in Galleria mellonella.

Injection of sterile latex beads into the hemocoel of last instar larvae of Galleria mellonella provoked a strong defense reaction. Cellular defense by hemocytes was followed by enhanced antibacterial activity in hemolymph. Latex-injected insects showed increased survival rates after a challenge injection with high doses of bacteria. Factors which stimulate the production of antibacterial activity could be demonstrated soon after injection by transfer of hemolymph from preinjected to untreated larvae. A large induction capacity in donor hemolymph was accompanied by a strong decrease in the total hemocyte count of free floating hemocytes, resulting from a decrease in number of plasmatocytes and granular cells, the cell types involved in the cellular defense against the injected latex beads. The results presented support the hypothesis that during cellular defense reactions, factors are released from the hemocytes which stimulate the production of antibacterial substances.

Further observations on the induction of immunity by hemolymph transfer in Galleria mellonella.

Humoral immune responses of Galleria mellonella larvae were provoked by transfer of hemolymph lysate supernatant preparations (HLS) from untreated donor larvae to recipient larvae. Eighteen hours after transfer the antibacterial activity in cell-free hemolymph of recipient larvae was as high as after injection of high dosages of living Enterobacter cloacae beta 12 bacteria. Filtration of HLS (FHLS; 0.2 microns filter) prior to injection enhanced its induction capacity. Injection of FHLS concentrates (> 30 < 100 kDa) provoked the same immune responses as injection of FHLS itself. Heat treatment did not destroy the induction capacity of FHLS. Hemolymph samples of larvae immunized by hemolymph transfer or by injection of bacteria showed the same characteristic protein pattern in SDS-PAGE. At least six new or enhanced bands were detected in hemolymph of immunized larvae. These bands were missing or very weak in hemolymph from untreated or saline injected larvae.

LPS (lipopolysaccharide)-activated immune responses in a hemocyte cell line from Estigmene acraea (Lepidoptera).

The suitability of the hemocyte cell line BTI-EA-1174-A from Estigmene acraea (Lepidoptera) to serve as a tool for studying insect immune reactions in vitro was investigated. Addition of bacterial lipopolysaccharides to the cultures caused enhanced phagocytosis of silica beads, as well as increased lysozyme activity in the cell culture supernatants. Addition of fungal beta 1,3-glucans did not result in any activation. The LPS-influenced (1 mg/mL) increase of phagocytic reactions against the silica beads was at its highest within 24 h after LPS-addition. Activated cells exhibited drastic changes in their morphology in connection with reduced cell numbers in the cultures but without increased mortality rates. LPS-dosages higher than 10 micrograms/mL LPS provoked significantly enhanced lysozyme activities. A maximal induction took place with 1 mg/mL LPS. The lysozyme activity started to rise 2 days after LPS-addition, further increase was observed up to the seventh day. The responsible protein was isolated from cell culture supernatants and N-terminally sequenced. The exact molecular mass was determined by mass spectrometry as 14.080 kDa. The amino acid sequence of the analysed portion revealed high sequence-similarity to the lysozymes of other lepidopteran insects as well as to hen egg lysozyme. Further results presented in this paper give indications for the existence of soluble molecules which are released by the cells and which enhance the LPS-triggered activation.

Gender differences and individual variation in the immune system of the scorpionfly Panorpa vulgaris (Insecta: Mecoptera).

From investigations of the vertebrate immune system gender specific differences in individual immunocompetence are well known. In general, females seem to possess more powerful immune systems than males. In invertebrates, the situation is much less clear. Therefore, we investigated the immune system of an invertebrate species, the scorpionfly Panorpa vulgaris. We found a high degree of individual variation in both traits studied, the lysozyme-like antibacterial activity of hemolymph and the capacity for in vitro phagocytosis of artificial particles. These two immune traits were positively correlated. As expected, hemolymph derived from females had higher lysozyme-like activity and hemocytes from females phagocytosed more particles. The difference in phagocytosis was mainly based on higher total hemocyte counts and higher proportions of phagocytically active cells in females, while the average number of ingested particles per active phagocyte was not significantly different. The observed gender differences are discussed in the context of reproductive strategies and parasite-mediated sexual selection.

Delineation of the catalytic domain of Clostridium difficile toxin B-10463 to an enzymatically active N-terminal 467 amino acid fragment.

In an attempt to directly approach the postulated toxic domain of Clostridium difficile's TcdB-10463, eight subclones of different size and locations in the N-terminal third of the toxin were generated. Expression of these toxin fragments was checked in Western blots and the enzymatic activity of the expressed proteins was analyzed by glucosylating Ras related small GTP-binding proteins. Two polypeptides of 875 aa (TcdBc1-3) and 557 aa (TcdBc1-H) glucosylated their targets Rho, Rac and Cdc42 with the same activity and specificity as the holotoxin. In comparison 516 aa (TcdBc1-N) and 467 aa (TcdBc1-A) protein fragments exhibited highly reduced activity, while Tcdc1 and TcdB2-3 (aa 1-243 and 244-890, respectively) were enzymatically inactive. Our results indicate that all structures involved in the catalysis are located at several different sites within the 557 aa fully active fragment. The shortest enzymatically still active protein covers aa 1-467 and obviously fulfils all minimal requirements for glucosylation. The data support the postulated three domain model of 'large clostridial cytotoxins'.

Regulation of nicotinic acetylcholine receptors by tyrosine kinases in the peripheral and central nervous system: same players, different roles.

Nicotinic acetylcholine receptors (nAChRs) exist in many subtypes and are found in the peripheral and central nervous system where they mediate or modulate synaptic transmission. We review how tyrosine phosphorylation and kinases regulate muscle and neuronal nAChRs. Interestingly, although some of the same kinase players interact with the various receptor subtypes, the functional consequences are different. While concerted action of MuSK, Abl- and Src-family kinases (SFKs) regulates the synaptic distribution of nAChRs at the neuromuscular junction, SFKs activate heteromeric neuronal nAChRs in adrenal chromaffin cells, thereby enhancing catecholamine secretion. In contrast, the activity of homomeric neuronal nAChRs, as found in the hippocampus, is negatively regulated by tyrosine phosphorylation and SFKs. It appears that tyrosine kinases provide the means to regulate all nAChRs; but the functional consequences, even those caused by the same kinase family, are specific for each receptor subtype and location.

Insect cell stimulation by LPS requires the activity of cell-released proteases.

The hemocyte line BTI-EA-1174-A from the lepidopteran insect Estigmene acraea responds to bacterial lipopolysaccharide (LPS) by an enhanced phagocytic reaction and a dose-dependent increase of lysozyme release [Wittwer et al., Dev Comp Immunol 21:323 (1997)]. This paper provides evidence for a strong proteolytic activity in cell culture supernatants occurring after addition of LPS (1 mg/ml). The proteolysis is caused by cell-released proteases and seems to be necessary for cell activation. Its inhibition by alpha 2-macroglobulin results in a dose-dependent reduction in cellular response strength. Phagocytic reactions, as well as lysozyme release, are lowered to about half in the presence of 0.0001 mg/ml alpha 2-macroglobulin. A nearly complete abolishment of activation was achieved with final concentrations of 1.0 mg/ml alpha 2-macroglobulin. The data presented allow us to conclude that the LPS-triggered proteolytic activity is an important part of the activation process; it occurs outside of the cells and delivers immune response activating factors.

Stimulation of NO synthase activity in the immune-competent lepidopteran Estigmene acraea hemocyte line.

In contrast to the vertebrate immune system, nearly nothing is known about the immunological role of nitric oxide (NO) in invertebrates. This study provides evidence of the presence of a NO synthase (NOS) activity in an immune-competent, macrophage-like insect hemocyte line, previously established from larvae of the lepidopteran insect Estigmene acraea. As proven by photometric determination of nitroblue tetrazolium reduction after cell fixation, the E. acraea cells possess NADPH diaphorase (NADPHd) activity. This NADPH diaphorase activity was NADPH dependent, not inhibitable by superoxide dismutase, influenced by extracellular addition of L-arginine, and inhibited in a dose-dependent manner by the specific NOS inhibitor Nomega-monomethyl-L-arginine. Furthermore, the NADPH diaphorase activity was stimulated within 30 min by the addition of insect pathogenic bacteria (Bacillus thuringiensis var. kurstaki, Photorhabdus luminescens), bacterial lipopolysaccharide, and silica beads. In activated E. acraea cell suspensions strongly increased amounts of L-citrulline and enhanced levels of total nitrite/nitrate (as NO derivates) can be determined. This is the first report on stimulable NOS activity in insect hemocytes.

Localization of injected apolipophorin III in vivo - new insights into the immune activation process directed by this protein.

A few years ago, it was shown that intrahemocoelic injection of the insect apolipoprotein apolipophorin III (apoLp-III) stimulates an immune response in larvae of the greater wax moth, Galleria mellonella. Since the mode of action of this activation process is unknown, we followed apoLp-III's pathway in the early phase of the immune-stimulating process, using biotin as a probe. Biotinylated apoLp-III was injected and localized using avidin-coupled horseradish peroxidase. The labeled protein was fully functional; the added amount of biotin per apoLp-III molecule used in this study only slightly decreased its ability to associate with phospholipase C-treated human low-density lipoprotein, as well as the immune-stimulating capability of apoLp-III.Gel electrophoresis with subsequent staining of biotin moieties and lipids revealed that apoLp-III undergoes lipid association in vivo within the first few minutes after injection. After two hours, no biotinylated apoLp-III was detectable in cell-free hemolymph. At this time, a subpopulation of hemocytes showed a distinct peroxidase staining. Control injections of biotinylated bovine serum albumin did not lead to similar results, giving evidence for the specificity of the phenomena observed. The results indicate that lipid association of apoLp-III occurs prior to endocytosis by immune-competent hemocytes, which is followed by the induction of a humoral immune response.

Successful parasitation of locusts by entomopathogenic nematodes is correlated with inhibition of insect phagocytes.

The entomopathogenic nematodes Heterorhabditis megidis and Steinernema feltiae turned out to be successful antagonists of the orthopteran insects Locusta migratoria and Schistocerca gregaria. The death rate of locusts maintained on nematode-inoculated sand was remarkably high. Even dosages as low as one nematode per cubic centimeter of sand killed approximately 50% of the locusts within 10 days. The impact of parasitation on locusts' immune defense was closely investigated for L. migratoria parasitized by H. megidis. Adult locusts died within 30-35 h after being fed with 50 infective H. megidis juveniles. Within the first 30 h after ingestion of the nematodes, locust hemolymph was assayed for alterations in the humoral and cellular defense components and for the presence of the nematode-associated Photorhabdus luminescens bacteria. Humoral defense was generally low without any correlation to the state of parasitation. There was no detectable activity against Escherichia coli and only little lysozyme-like activity against Micrococcus luteus. In contrast, cellular defense components were strongly influenced by parasitation. Most interestingly, the phagocytic capacity of the hemocytes was already hampered 12 h after oral application of the nematodes, whereas considerable hemocyte death occurred not earlier than 24 h after feeding. The nematode-associated bacteria could be detected in hemolymph of some of the nematode-fed locusts as early as 3 h after feeding and in all hemolymph samples after 24 h. Supernatants from isolated P. luminescens cultures were able to inhibit the L. migratoria phagocytes in vitro; thus the successful parasitation appears to be dependent on an inhibition by bacteria-released compounds rather than on overloading or simply killing of the phagocytic active hemocytes.

[Factors influencing the corrosion of amalgams]. / Einflussgrössen auf das Korrosionsverhalten von Amalgamen.

In a comparative corrosion test specimen were produced from eleven different amalgams using two different plugging pressures and were exposed to two different corrosive solutions. As was to be expected containing amalgams corroded the most. A more accurate differentiation between the individual non gamma 2 amalgams and the two different plugging pressures was only possible with the stronger corrosive solution. Therefore this solution seemed to be better suited for a standardized corrosion test according to DIN 13904, part 2. The greater plugging pressure inhibits corrosion especially in badly corroding amalgams. This seems to be due to the limited diffusion which is the result of a smaller pore size.

Lipophorin of lower density is formed during immune responses in the lepidopteran insect Galleria mellonella.

Injection of heat-killed bacteria into larvae of the greater wax moth Galleria mellonella is followed by changes in lipoprotein composition in the hemolymph. Density gradient centrifugation experiments revealed that within the first four hours after injection, a part of larval lipoprotein, high-density lipophorin (HDLp), was converted into a lipoprotein of lower density. SDS-polyacrylamide gel electrophoresis analysis of the gradient fractions and sequencing of protein fragments, established that the exchangeable apolipoprotein apolipophorin III (apoLp-III), a potent immune-activator, was associated with this newly formed lipophorin. To investigate further the influence of lipophorin-associated apoLp-III on immune-related reactions, we performed in vitro studies with isolated hemocytes from G. mellonella and lipophorins from the sphinx moth Manduca sexta, as a natural source of high amounts of low-density lipophorin (LDLp) and HDLp. The hemocytes were activated to form superoxide radicals upon incubation with LDLp, but not with HDLp. Fluorescence-labeled LDLp was specifically taken up by granular cells. This process was inhibited by adding an excess of unlabeled LDLp, but not by HDLp. We hypothesize that larval lipophorin formed in vivo is an endogenous signal for immune activation, specifically mediated by the binding of lipid-associated apoLp-III to hemocyte membrane receptors.

Primary structure of apolipophorin-III from the greater wax moth, Galleria mellonella.

The complete amino acid sequence of apolipophorin-III (apoLp-III), a lipid-binding hemolymph protein from the greater wax moth, Galleria mellonella, was determined by protein sequencing. The mature protein consists of 163 amino acid residues forming a protein of 18,075.5 Da. Its sequence is similar to apoLp-III from other Lepidopteran species, but remarkably different from the apoLp-IIIs of insects from other orders. As shown by mass spectrometric analysis, the protein carries no modifications. Thus, all of its known physiological functions, including its recently discovered immune response-stimulating activity, must reside in the protein itself.