Search

Abstract Tumor-infiltrating lymphocytes are considered clinically beneficial in breast cancer, but the significance of natural killer (NK) cells is less well characterized. As increasing evidence has demonstrated that the spatial organization of immune cells in tumor microenvironments is a significant parameter for impacting disease progression as well as therapeutic responses, an improved understanding of tumor-infiltrating NK cells and their location within tumor contextures is needed to improve the design of effective NK cell-based therapies. In this study, we developed a multiplex immunohistochemistry (mIHC) antibody panel designed to quantitatively interrogate leukocyte lineages, focusing on NK cells and their phenotypes, in two independent breast cancer patient cohorts (n = 26 and n = 30). Owing to the clinical evidence supporting a significant role for NK cells in HER2+ breast cancer in mediating responses to Trastuzumab, we further evaluated HER2- and HER2+ specimens separately. Consistent with literature, we found that CD3+ T cells were the dominant leukocyte subset across breast cancer specimens. In comparison, NK cells, identified by CD56 or NKp46 expression, were scarce in all specimens with low granzyme B expression indicating reduced cytotoxic functionality. Whereas NK cell density and phenotype did not appear to be influenced by HER2 status, spatial analysis revealed distinct NK cells phenotypes regarding their proximity to neoplastic tumor cells that associated with HER2 status. Spatial cellular neighborhood analysis revealed multiple unique neighborhood compositions surrounding NK cells, where NK cells from HER2- tumors were more frequently found proximal to neoplastic tumor cells, whereas NK cells from HER2+ tumors were instead more frequently found proximal to CD3+ T cells. This study establishes the utility of quantitative mIHC to evaluate NK cells at the single-cell spatial proteomics level and illustrates how spatial characteristics of NK cell neighborhoods vary within the context of HER2- and HER2+ breast cancers.

Background: Employment relations trust (ERT) is related to compliance, fairness, good faith and benevolence in such relations. Benevolence is not a legal obligation in employment relations and will typically be displayed voluntarily. Gaps in the literature suggested a need for a valid and reliable theory of employment relations benevolence and its relationship with trust in such relations. Aim: This study was undertaken to investigate the relationship between benevolence and the development of trust in employment relations. Setting: The study was undertaken in the Gauteng province of the Republic of South Africa. Methods: An exploratory quantitative research method was implemented to test two hypotheses emanating from a literature review. A questionnaire survey facilitated the collection of data from 83 purposefully sampled voluntary respondents, who were ordinary employees, managers and academics. Statistical analysis was done to test the validity of two hypotheses. Results: Employment relations trust can be expected to develop through four distinct, overlapping and relatively predictable sequential stages, namely compliance, fairness, good faith and benevolence. Benevolence can be assumed to be the last stage in this development process. Conclusion: Benevolence can be regarded as the erratic pinnacle of a four levelled theoretical hierarchy of ERT. Benevolence can occur before, during or after displays of compliance, fairness or good faith in employment relations. Contribution: This theory of ERT provides valid and reliable foundations for dealing with and researching a variety of aspects related to benevolence and trust in employment relations.

Infusion of ex vivo expanded and cytokine-activated natural killer (NK) cells is a promising alternative way to treat multiple myeloma (MM). However, the tumor microenvironment (TME) may suppress their function. While reduced glucose availability is a TME hallmark of many solid tumors, glucose levels within the TME of hematological malignancies residing in the bone marrow (BM) remain unknown. Here, we measured glucose levels in the BM of MM patients and tested the effect of different glucose levels on NK cells. BM glucose levels were measured using a biochemical analyzer. Compared to the normal range of blood glucose, BM glucose levels were lower in 6 of 9 patients (479-1231 mg/L; mean=731.8 mg/L). The effect of different glucose levels on NK cell cytotoxicity was tested in 4-hour cytotoxicity assays with tumor cells. 500 mg/L glucose (representing low range of MM BM) during the 4-hour cytotoxicity assay did not negatively affect cytotoxicity of activated NK cells, while higher glucose concentrations (4000 mg/L) diminished NK cell cytotoxicity. Since clinical application of NK cell therapy might require ex vivo expansion, expanded NK cells were exposed to a range of glucose concentrations from 500-4000 mg/L for a longer period (4 days). This did not reduce cytotoxicity or IFN-γ secretion nor affected their phenotypic profile. In summary, low glucose concentrations, as found in BM of MM patients, by itself did not compromise the anti-tumor potential of IL-2 activated NK cells in vitro. Although follow up studies in models with a more complex TME would be relevant, our data suggest that highly activated NK cells could be used to target tumors with a reduced glucose environment.

Background: Primary labour relationships (PLRs) occur within supervisory relationships. Previous studies confirmed that compliance, fairness, good faith and trust were interrelated facilitators of positive perceptions of primary labour relationship quality. Many researchers regard trust as a primary requirement for effective implementation of formal and psychological employment contracts. Aim: This study investigated the nature and direction of relationships between subordinate employee perceptions of the levels of compliance, fairness and good faith in PLRs and levels of trust in PLRs. Setting: Two interviewers adopted snowball sampling approaches to conduct structured interviews with 68 subordinate employees residing in Gauteng, South Africa. Methods: The researcher adopted a mixed-method research methodology that included a thorough literature review, development of a structured interview, interviewing 68 voluntary participants and statistical analysis of data. Results: Confident conclusions were drawn and discussed, and related limitations were explained. Specific recommendations for further research into the relationships and dynamics of trust-related phenomena in PLRs were made. Conclusion: It was confidently concluded that literature and empirical findings, jointly and separately, provided ample evidence of positive relationships between subordinate employee perceptions of the levels of trust and their perceptions of the levels of compliance, fairness and good faith in PLRs. Accordingly, it can be confidently expected that lower levels of trust will be related to lower levels of perceived compliance, fairness and good faith in PLRs, and higher levels of trust will be related to higher levels of perceived compliance, fairness and good faith in PLRs. Causality was not investigated in this study.

Natural killer (NK) cells are attractive candidates for allogeneic cell-based immunotherapy due to their potent antitumor effector function and good safety profile. NK cells express killer immunoglobulin-like receptors (KIRs) and the NKG2A receptor important for NK cells education as well as providing inhibitory signals upon encountering HLA-expressing target cells. Multiple myeloma (MM) is an example of a tumor expressing relatively high levels of HLA molecules. In this review, we discuss the functional relevance of inhibitory KIRs and NKG2A for NK cells anti-MM response and strategies to lower these inhibitory signaling to enhance clinical efficacy of allogeneic NK cells in MM.

Natural killer (NK) cell-based immunotherapy is a promising therapy for cancer patients. Inhibitory killer immunoglobulin-like receptors (KIRs) and NKG2A are required for NK cell licensing, but can also inhibit NK cell effector function. Upon reconstitution in a stem cell transplantation setting or after ex vivo NK expansion with IL-2, NKG2A is expressed on a large percentage of NK cells. Since the functional consequences of NKG2A co-expression for activated NK cells are not well known, we compared NKG2A+ vs NKG2A− NK cell subsets in response to K562 cells, multiple myeloma (MM) cell lines and primary MM cells. NK cells were isolated from healthy donors (HLA-C1+C2+Bw4+) and activated overnight with 1,000 U/ml IL-2. NK cell degranulation in subsets expressing KIRs and/or NKG2A was assessed at 21 or 0.6% O2. Activated NKG2A+ NK cell subsets degranulated more vigorously than NKG2A− subsets both at 21 and 0.6% O2. This was irrespective of the presence of KIR and occurred in response to HLA-deficient K562 cells as well as HLA competent, lowly expressing HLA-E MM cell lines. In response to primary MM cells, no inhibitory effects of NKG2A were observed, and NKG2A blockade did not enhance degranulation of NKG2A+ subsets. KIR− NK cells expressing NKG2A degranulated less than their NKG2A− counterparts in response to MM cells having high levels of peptide-induced membrane HLA-E, suggesting that high surface HLA-E levels are required for NKG2A to inhibit activated NK cells. Addition of daratumumab, an anti-CD38 to trigger antibody-dependent cell-mediated cytotoxicity, improved the anti-MM response for all subsets and degranulation of the KIR−NKG2A− “unlicensed” subset was comparable to KIR+ or NKG2A+ licensed subsets. This demonstrates that with potent activation, all subsets can contribute to tumor clearance. Additionally, subsets expressing KIRs mismatched with the HLA ligands on the target cell had the highest level of activation in response to MM cell lines as well as against primary MM. Our current study demonstrated that if NK cells are sufficiently activated, e.g., via cytokine or antibody activation, the (co-)expression of NKG2A receptor may not necessarily be a disadvantage for NK cell-based therapy.

Background: A typology of desirable social conditions in supervisory relationships suggested that such conditions may also be desirable in other forms of labour relationships. A literature review confirmed that trust, compliance, fairness and good faith can be confidently regarded as universally desirable social conditions in all forms of individual or collective labour relationships between employers and employees. Aim: The purpose of this study was to determine if primary labour relationship quality (PLQ) can be confidently conceptualised as a social construct that strongly relates to the perceived levels of compliance, fairness and good faith in supervisory or primary labour relationships. Setting: A combination of random and convenience sampling approaches was implemented to collect PLQ related data from 454 voluntary respondents, who were subordinate employees in the Tshwane region. Methods: A quantitative research methodology was adopted. This included conceptual definition of the PLQ construct, objective measurement of PLQ levels of voluntary respondents in an adequately sized sample, factor analysis and testing for relationships and differences in means between variables. Results: Data analysis results confirmed that it can be confidently concluded that the conceptual definition of PLQ was valid, and that positive PLQ perceptions of subordinate employees were significantly related to at least two other forms of desirable organisational outcomes. Conclusion: PLQ perceptions can be confidently defined as a distinct subjective quality estimate that is assimilated from unique expectations and perceptions of the levels of compliance, fairness, good faith and trust that a supervisor displays in a labour relationship with an immediate subordinate.

High salt in diet elevates blood pressure in man. Berries and fruits are regarded beneficial in antagonizing hypertension and related vascular complications. We aimed to test whether lingonberry juice shows beneficial and anti-inflammatory effects in salt induced hypertension rat model. Male Wistar–Kyoto rats were fed with 8% sodium chloride enriched pellets for 10 weeks having tap water or diluted lingonberry juice as drinking fluid. Blood pressure was recorded weekly and mesenteric artery functions tested ex vivo. Urine was collected in metabolic caging. Salt loading had only minor effects on blood pressure or endothelial function. Increased urine excretion of 8-isoprostane, cGMP and albumin reflected general or renal inflammation. Elevated expression (mRNA) of proinflammatory COX-2 by salt was normalized by lingonberry. Salt increased kidney, heart and left ventricle masses and changed serum chloride, alkaline phosphatase, albumin and lipid concentrations. Taken together the results show that l0 weeks’ high-salt diet impaired kidney function of young rats without clear effect on blood pressure or vascular function. Lingonberry juice moderately reduced biomarkers of low-grade inflammation probably due to its high polyphenol concentrations.

Lingonberries (Vaccinium vitis-idaea) are rich in polyphenols, such as proanthocyanidins, anthocyanins, flavonols and flavanols. Polyphenol-rich foods affect vascular health. We previously described improved endothelium-dependent vascular function as well as anti-inflammatory and anti-atherothrombotic effects in spontaneously hypertensive rats (SHR) fed with lingonberry juice. In the present study, we investigated the effects of lingonberry juice dose on blood pressure, vascular function and vascular inflammation in SHR in an 8 weeks’ study. Diluted (1:5) cold-compressed lingonberry juice was given as drinking fluid ad libitum to 5 week old SHR for 8 weeks. Control group (SHR) and normotensive reference group (Wistar-Kyoto) received tap water. Systolic blood pressure (SBP) was monitored weekly. Function of mesenteric artery rings was assessed in standard organ-bath chambers after 8 weeks. Biochemical and clinical chemistry variables were measured from plasma and urine. Lingonberry juice lowered SBP of SHR (p = 0.007). Endothelium-dependent vascular relaxation was not improved. Lingonberry treatment slightly affected plasma inflammatory markers (reduction of NOx and sICAM-1) and clinical chemistry variables (decreased alkaline phosphatase and increased chloride and calcium levels). In conclusion, Lingonberry juice at small concentrations lowers blood pressure in a long-term treatment in SHR.

Flavonoids and other phenolic compounds affect low-grade inflammation related to cardiovascular diseases among other positive health effects. Cardioprotective actions are mainly due to enhanced endothelial function and production of nitric oxide (NO). We investigated vascular anti-inflammatory effects of cranberry (Vaccinium oxycoccos), lingonberry (Vaccinium vitis-idaea) and blackcurrant (Ribes nigrum) juices given as drinking fluid ad libitum to spontaneously hypertensive rats (SHR), a widely used model of human hypertension, in an 8 week intervention study. The animals were sacrificed, the aortas cleaned and RNA was extracted. cDNA was prepared for real-time PCR and blood was collected for biochemical analyses. The mRNA expressions of angiotensin-converting enzyme 1 (ACE1), cyclooxygenase 2 (COX2), monocyte chemoattractant protein 1 (MCP1) and P-selectin were significantly reduced in the cranberry and lingonberry groups. These findings suggest that cranberry and lingonberry cold-compressed juices have anti-inflammatory and anti-atherothrombotic actions in long-term treatment of SHR.

Phenolic compounds of berries, fruits and vegetables affect vascular health. Endothelium-dependent vasodilatation has an important role in blood pressure development by controlling the vascular tone. Endothelial cells produce and release various relaxing and contracting factors, like nitric oxide (NO), cyclo-oxygenase (COX)-derived prostanoids and endothelium-derived hyperpolarizing factor (EDHF). The purpose of this study was to investigate whether 8 weeks treatment with Finnish berry juices, cranberry (Vaccinium oxycoccos), lingonberry (Vaccinium vitis-idaea) and blackcurrant (Ribes nigrum) affects blood pressure and vascular function of spontaneously hypertensive rats (SHR). After 8 weeks treatment the mesenteric arteries of the rats were taken for vascular reactivity studies. Lingonberry treatment normalized the impaired endothelium-dependent relaxation seen in the cranberry, blackcurrant and control rats. In the arteries of lingonberry treated rats the relaxation was partly due to NO, but also dependent on EDHF. It can be concluded that long-term lingonberry juice treatment improves endothelium-dependent vasodilatation of SHR.

Stable isotope abundances convey valuable information about plant physiological processes and underlying environmental controls. Central gaps in our mechanistic understanding of hydrogen isotope abundances impede their widespread application within the plant and biogeosciences. To address these gaps, we analysed intramolecular deuterium abundances in glucose of Pinus nigra extracted from an annually resolved tree-ring series (1961-1995). We found fractionation signals (i.e. temporal variability in deuterium abundance) at glucose H 1 and H 2 introduced by closely related metabolic processes. Regression analysis indicates that these signals (and thus metabolism) respond to drought and atmospheric CO 2 concentration beyond a response change point. They explain ≈ 60% of the whole-molecule deuterium variability. Altered metabolism is associated with below-average yet not exceptionally low growth. We propose the signals are introduced at the leaf level by changes in sucrose-to-starch carbon partitioning and anaplerotic carbon flux into the Calvin-Benson cycle. In conclusion, metabolism can be the main driver of hydrogen isotope variation in plant glucose., (© 2022 The Authors New Phytologist © 2022 New Phytologist Foundation.)

Natural peatlands contribute significantly to global carbon sequestration and storage of biomass, most of which derives from Sphagnum peat mosses. Atmospheric CO 2 levels have increased dramatically during the twentieth century, from 280 to > 400 ppm, which has affected plant carbon dynamics. Net carbon assimilation is strongly reduced by photorespiration, a process that depends on the CO 2 to O 2 ratio. Here we investigate the response of the photorespiration to photosynthesis ratio in Sphagnum mosses to recent CO 2 increases by comparing deuterium isotopomers of historical and contemporary Sphagnum tissues collected from 36 peat cores from five continents. Rising CO 2 levels generally suppressed photorespiration relative to photosynthesis but the magnitude of suppression depended on the current water table depth. By estimating the changes in water table depth, temperature, and precipitation during the twentieth century, we excluded potential effects of these climate parameters on the observed isotopomer responses. Further, we showed that the photorespiration to photosynthesis ratio varied between Sphagnum subgenera, indicating differences in their photosynthetic capacity. The global suppression of photorespiration in Sphagnum suggests an increased net primary production potential in response to the ongoing rise in atmospheric CO 2 , in particular for mire structures with intermediate water table depths., (© 2021. The Author(s).)

Measurements of carbon isotope contents of plant organic matter provide important information in diverse fields such as plant breeding, ecophysiology, biogeochemistry and paleoclimatology. They are currently based on 13 C/ 12 C ratios of specific, whole metabolites, but we show here that intramolecular ratios provide higher resolution information. In the glucose units of tree-ring cellulose of 12 tree species, we detected large differences in 13 C/ 12 C ratios (>10‰) among carbon atoms, which provide isotopically distinct inputs to major global C pools, including wood and soil organic matter. Thus, considering position-specific differences can improve characterisation of soil-to-atmosphere carbon fluxes and soil metabolism. In a Pinus nigra tree-ring archive formed from 1961 to 1995, we found novel 13 C signals, and show that intramolecular analysis enables more comprehensive and precise signal extraction from tree rings, and thus higher resolution reconstruction of plants' responses to climate change. Moreover, we propose an ecophysiological mechanism for the introduction of a 13 C signal, which links an environmental shift to the triggered metabolic shift and its intramolecular 13 C signature. In conclusion, intramolecular 13 C analyses can provide valuable new information about long-term metabolic dynamics for numerous applications.

Gastrointestinal stromal tumors (GISTs) predominantly harbor activating mutations in the receptor tyrosine kinase KIT. To genetically dissect in vivo the requirement of different signal transduction pathways emanating from KIT for tumorigenesis, the oncogenic Kit V558Δ mutation was combined with point mutations abrogating specific phosphorylation sites on KIT. Compared with single-mutant Kit V558Δ/+ mice, double-mutant Kit V558Δ;Y567F/Y567F knock-in mice lacking the SRC family kinase-binding site on KIT (pY567) exhibited attenuated MAPK signaling and tumor growth. Surprisingly, abrogation of the PI3K-binding site (pY719) in Kit V558Δ;Y719F/Y719F mice prevented GIST development, although the interstitial cells of Cajal (ICC), the cells of origin of GIST, were normal. Pharmacologic inhibition of the PI3K pathway in tumor-bearing Kit V558Δ/+ mice with the dual PI3K/mTOR inhibitor voxtalisib, the pan-PI3K inhibitor pilaralisib, and the PI3K-alpha-restricted inhibitor alpelisib each diminished tumor proliferation. The addition of the MEK inhibitor PD-325901 or binimetinib further decreased downstream KIT signaling. Moreover, combining PI3K and MEK inhibition was effective against imatinib-resistant Kit V558Δ;T669I/+ tumors., Competing Interests: The authors declare no conflict of interest.

Terrestrial vegetation currently absorbs approximately a third of anthropogenic CO2 emissions, mitigating the rise of atmospheric CO2. However, terrestrial net primary production is highly sensitive to atmospheric CO2 levels and associated climatic changes. In C3 plants, which dominate terrestrial vegetation, net photosynthesis depends on the ratio between photorespiration and gross photosynthesis. This metabolic flux ratio depends strongly on CO2 levels, but changes in this ratio over the past CO2 rise have not been analyzed experimentally. Combining CO2 manipulation experiments and deuterium NMR, we first establish that the intramolecular deuterium distribution (deuterium isotopomers) of photosynthetic C3 glucose contains a signal of the photorespiration/photosynthesis ratio. By tracing this isotopomer signal in herbarium samples of natural C3 vascular plant species, crops, and a Sphagnum moss species, we detect a consistent reduction in the photorespiration/photosynthesis ratio in response to the ∼100-ppm CO2 increase between ∼1900 and 2013. No difference was detected in the isotopomer trends between beet sugar samples covering the 20th century and CO2 manipulation experiments, suggesting that photosynthetic metabolism in sugar beet has not acclimated to increasing CO2 over >100 y. This provides observational evidence that the reduction of the photorespiration/photosynthesis ratio was ca. 25%. The Sphagnum results are consistent with the observed positive correlations between peat accumulation rates and photosynthetic rates over the Northern Hemisphere. Our results establish that isotopomers of plant archives contain metabolic information covering centuries. Our data provide direct quantitative information on the "CO2 fertilization" effect over decades, thus addressing a major uncertainty in Earth system models.

Background and Purpose: Working memory impairment is among the earliest signs of cognitive decline in Alzheimer's disease (AD) and mild cognitive impairment (MCI). We aimed to study the functional and structural substrate of working memory impairment in early AD dementia and MCI., Methods: We studied a group of 12 MCI and AD subjects compared to 12 age- and gender-matched healthy elderly controls using diffusion tensor imaging (DTI), and functional magnetic resonance imaging (fMRI) during a 2-back versus 1-back letter recognition task. We performed a three-way image fusion analysis with joint independent component analysis of cortical activation during working memory, and DTI derived measures of fractional anisotropy (FA) and the mode of anisotropy., Results: We found significant hypoactivation in posterior brain areas and relative hyperactivation in anterior brain areas during working memory in AD/MCI subjects compared to controls. Corresponding independent components from DTI data revealed reduced FA and reduced mode of anisotropy in intracortical projecting fiber tracts with posterior predominance and increased FA and increased mode along the corticospinal tract in AD/MCI compared to controls., Conclusions: Our findings suggest that impairments of structural fiber tract integrity accompany breakdown of posterior and relatively preserved anterior cortical activation during working memory performance in MCI/AD subjects., (Copyright © 2014 by the American Society of Neuroimaging.)

Mitigation of N-nitrosodimethylamine (NDMA) and other hazardous water disinfection byproducts (DBP) is currently hampered by a limited understanding of DBP formation mechanisms. Because variations of the stable isotope composition of NDMA can potentially reveal reaction pathways and precursor compounds, we developed a method for the compound-specific isotope analysis (CSIA) of (13)C/(12)C, (15)N/(14)N, and (2)H/(1)H ratios of NDMA by gas chromatography coupled to isotope ratio mass spectrometry (GC/IRMS). Method quantification limits for the accurate isotope analysis of NDMA, N-nitrosodiethyl-, -dipropyl-, and -dibutylamine as well as N-nitrosopyrrolidine were between 0.18 to 0.60 nmol C, 0.40 to 0.80 nmol N, and 2.2 to 5.8 nmol H injected on column. Coupling solid phase extraction (SPE) to GC/IRMS enabled the precise quantification of C, N, and H isotope ratios of NDMA in aqueous samples at concentrations of 0.6 μM (45 μg L(-1)). We validated the proposed method with a laboratory experiment, in which NDMA was formed with stoichiometric yield (97 ± 4%) through chloramination of the pharmaceutical ranitidine (3 μM). δ(13)C and δ(2)H values of NDMA remained constant during NDMA formation while its δ(15)N increased due to a reaction at a N atom in the rate-limiting step of NDMA formation. The δ(2)H value of NDMA determined by SPE-GC/IRMS also corresponded well to the δ(2)H value of the N(CH3)2-group of ranitidine measured by quantitative deuterium nuclear magnetic resonance spectroscopy. This observation implies that the N(CH3)2-moiety of ranitidine is transferred to NDMA without being chemically altered and illustrates the accuracy of the proposed method.

Background: Research suggests generally impaired cognitive control functions in working memory (WM) processes in amnestic mild cognitive impairment (MCI) and incipient Alzheimer's disease (AD). Little is known how emotional salience of task-irrelevant stimuli may modulate cognitive control of WM performance and neurofunctional activation in MCI and AD individuals., Objective: We investigated the impact of emotional task-irrelevant visual stimuli on cortical activation during verbal WM., Methods: Twelve AD/MCI individuals and 12 age-matched healthy individuals performed a verbal WM (nback-) task with task-irrelevant emotionally neutral and emotionally negative background pictures during fMRI measurement., Results: AD/MCI individuals showed decreased WM performance compared with controls; both AD/MCI and control groups reacted slower during presentation of negative pictures, regardless of WM difficulty. The AD/MCI group showed increased activation in the left hemispheric prefrontal network, higher amygdala and less cerebellar activation with increasing WM task difficulty compared to healthy controls. Correlation analysis between neurofunctional activation and WM performance revealed a negative correlation between task sensitivity and activation in the dorsal anterior cingulum for the healthy controls but not for the AD/MCI group., Conclusion: Our data suggest compensatory activation in prefrontal cortex and amygdala, but also dysfunctional inhibition of distracting information in the AD/MCI group during higher WM task difficulty. Additionally, attentional processes affecting the correlation between WM performance and neurofunctional activation seem to be different between incipient AD and healthy aging.

The persistent organic pollutant DDT (1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane) is still indispensable in the fight against malaria, although DDT and related compounds pose toxicological hazards. Technical DDT contains the dichloro congener DDD (1-chloro-4-[2,2-dichloro-1-(4-chlorophenyl)ethyl]benzene) as by-product, but DDD is also formed by reductive degradation of DDT in the environment. To differentiate between DDD formation pathways, we applied deuterium NMR spectroscopy to measure intramolecular deuterium distributions (2H isotopomer abundances) of DDT and DDD. DDD formed in the technical DDT synthesis was strongly deuterium-enriched at one intramolecular position, which we traced back to 2H/1H fractionation of a chlorination step in the technical synthesis. In contrast, DDD formed by reductive degradation was strongly depleted at the same position, which was due to the incorporation of 2H-depleted hydride equivalents during reductive degradation. Thus, intramolecular isotope distributions give mechanistic information on reaction pathways, and explain a puzzling difference in the whole-molecule 2H/1H ratio between DDT and DDD. In general, our results highlight that intramolecular isotope distributions are essential to interpret whole-molecule isotope ratios. Intramolecular isotope information allows distinguishing pathways of DDD formation, which is important to identify polluters or to assess DDT turnover in the environment. Because intramolecular isotope data directly reflect isotope fractionation of individual chemical reactions, they are broadly applicable to elucidate transformation pathways of small bioactive molecules in chemistry, physiology and environmental science.

Phospholipids (PLs) comprise an important lipid class in food because of their technological use as emulsifiers and their nutritional value. This study used one-dimensional (31)P NMR and two-dimensional (2D) (31)P,(1)H COSY NMR spectroscopy for the determination of the PL composition of cheese and fish after liquid-liquid enrichment. This extraction step enabled the identification of 10 PLs in cheese and 9 PLs in fish by 2D (31)P,(1)H NMR. Variations in the (31)P shifts indicated differences in the fatty acids attached to the individual PLs. The total PL content in cheese fat and fish oil ranged from 0.3 to 0.4% and from 5 to 12%, respectively. Phosphatidylcholine was the most prominent PL in both matrices (up to 65%). Minor PLs (limit of detection = 4 nmol, i.e. 500 μL of an 8 μM solution) were identified in forms of phosphatidic acid, lysophosphatidic acid, and phosphatidylglycerol. Specific cross couplings and (1)H fine structures in the 2D (31)P,(1)H NMR spectra proved to be valuable for the assignment and verification of known and uncommon PLs in the samples.

Kit receptor-activating mutations are critical in the pathogenesis of gastrointestinal stromal tumors (GIST). We investigated mechanisms of oncogenic Kit signaling and the consequences of therapeutic intervention in a mouse model of human GIST. Treatment of GIST mice with imatinib decreased cell proliferation and increased apoptosis in the tumor. Analysis of tumor tissue from imatinib-treated mice showed diminished phosphatidylinositol 3-kinase (PI3-kinase) and mammalian target of rapamycin (mTOR) signaling suggesting that oncogenic Kit signaling critically contributes to the translational response in GIST. Treatment with RAD001 (everolimus), an mTOR inhibitor, diminished the translational response and cell proliferation in tumor lesions, pointing to mTOR inhibition as a therapeutic approach for imatinib-resistant GIST. Analysis of RNA expression profiles in GIST lesions with and without imatinib treatment showed changes in expression of IFN-inducible genes and cell cycle regulators. These results convincingly show that KitV558Delta/+ mice represent a unique faithful mouse model of human familial GIST, and they demonstrate the utility of these mice for preclinical investigations and to elucidate oncogenic signaling mechanisms by using genetic approaches and targeted pharmacological intervention.

The La protein is a multifunctional RNA-binding protein and has also been suggested to be involved in the stabilization of hepatitis B virus (HBV) RNA. Here we demonstrate that antibodies against the human La protein specifically precipitate HBV RNA from HBV ribonucleoprotein-containing mammalian cell extracts, providing evidence for the association between human La and HBV RNA. Moreover, we report that the turnover of HBV RNA depends on structural features and less on the primary sequence of the La-binding site on the viral RNA. In addition we show that the interaction between human La and HBV RNA in vitro is modulated by accessory factor(s) in a phosphorylation-dependent manner. Taken together these data indicate that both structural features, the composition of La/HBV ribonucleoprotein particles as well as interacting cellular factors, are critical determinants in the regulation of the stability of the HBV RNA.

Background: Previous studies have shown a 10-fold discrepancy of self-reported food-induced symptoms and physician-diagnosed food hypersensitivity. Little information is available on the prevalence of food hypersensitivity in unselected paediatric populations. No data were available for German children., Objective: To study the perception of food-induced symptoms in the paediatric population, to investigate the allergens accused, to objectify patients' reports, and to identify subgroups at risk of having food-induced allergy (FA) or non-allergic food hypersensitivity (NAFH) reactions., Methods: This paper presents the data of the paediatric group (0-17 years) of a representative, randomly sampled, cross-sectional population-based survey studying 13 300 inhabitants of the German capital city Berlin regarding food-related symptoms. Instruments included mailed questionnaires, structured telephone interviews, physical examination, skin-prick tests, specific serum IgE and standardized, controlled and blinded oral food challenges., Results: Two thousand three hundred and fifty-four individuals were contacted by mailed questionnaire, 739 (31.4%) responses could be fully evaluated. Four hundred and fifty-five (61.5%) participants reported symptoms related to food ingestion, 284 (38.4%) affirmed reproducible symptoms in the standardized telephone interview. One hundred and eighty-four (24.8%) individuals were fully examined. Reproducible symptoms to food were found in 31 (4.2%) children and adolescents: 26 (3.5%) showed symptoms of FA and five (0.7%) of NAFH. The oral allergy syndrome was most often observed. Foods most commonly identified by oral challenges were apple, hazelnut, soy, kiwi, carrot and wheat., Conclusion: The perception of food-related symptoms is common among children and adolescents from the general population. Self-reports could be confirmed in around one out of 10 individuals, still resulting in 4.2% of proven clinical symptoms. However, most reactions were mild and mainly because of pollen-associated FA, while NAFH reactions were less common. Severe IgE-mediated FA was observed in individuals with pre-existing atopic disease, who should be fully investigated for clinically relevant FA.

The Kit receptor functions in hematopoiesis, lymphocyte development, gastrointestinal tract motility, melanogenesis, and gametogenesis. To investigate the roles of different Kit signaling pathways in vivo, we have generated knock-in mice in which docking sites for PI 3-kinase (KitY719) or Src kinase (KitY567) have been mutated. Whereas steady-state hematopoiesis is normal in KitY719F/Y719F and KitY567F/Y567F mice, lymphopoiesis is affected differentially. The KitY567F mutation, but not the KitY719F mutation, blocks pro T cell and pro B cell development in an age-dependent manner. Thus, the Src family kinase, but not the PI 3-kinase docking site in Kit, mediates a critical signal for lymphocyte development. In agreement with these results, treatment of normal mice with the Kit tyrosine kinase inhibitor imatinib (Gleevec) leads to deficits in pro T and pro B cell development, similar to those seen in KitY567F/Y567F and KitW/W mice. The two mutations do not affect embryonic gametogenesis but the KitY719F mutation blocks spermatogenesis at the spermatogonial stages and in contrast the KitY567F mutation does not affect this process. Therefore, Kit-mediated PI 3-kinase signaling and Src kinase family signaling is highly specific for different cellular contexts in vivo.

Objective: A population study was performed to identify the prevalence of all kinds of adverse reactions to food., Methods: In a representative cross-sectional survey performed in 1999 and 2000 in Berlin, 13 300 inhabitants of all ages were addressed by questionnaire. This questionnaire was answered by 4093 persons. All respondents mentioning any sign of food intolerance or the existence of allergic diseases (n = 2298) were followed up by telephone and, in case food intolerance could not be ruled out by patient history, were invited to attend to the clinic for personal investigation including double-blind, placebo-controlled food challenge tests (DBPCFC)., Results: The self-reported lifetime prevalence of any adverse reaction to food in the Berlin population (mean age 41 years) was 34.9%. Eight hundred and fourteen individuals were personally investigated according to the guidelines. The point prevalence of adverse reactions to food confirmed by DBPCFC tests in the Berlin population as a mean of all age groups was 3.6% (95% confidence interval [3.0-4.2%]) and 3.7% in the adult population (18-79 years, 95% confidence interval [3.1-4.4.%]). Two and a half percent were IgE-mediated and 1.1% non-IgE-mediated, females were more frequently affected (60.6%). Based on a statistical comparison with available data of adults from the nationwide German Health Survey from 1998, adverse reactions to food in the adult population of Germany (age 18-79) were calculated with 2.6% [2.1-3.2%])., Conclusions: The study gives for the first time information about the point prevalence of both immunological and nonimmunological adverse reactions to food and underlines the relevance of this issue in public health. The data also show that an individualized stepwise approach including provocation tests is mandatory to confirm the diagnosis.

Oncogenic Kit mutations are found in somatic gastrointestinal (GI) stromal tumors (GISTs) and mastocytosis. A mouse model for the study of constitutive activation of Kit in oncogenesis has been produced by a knock-in strategy introducing a Kit exon 11-activating mutation into the mouse genome based on a mutation found in a case of human familial GIST syndrome. Heterozygous mutant KitV558Delta/+ mice develop symptoms of disease and eventually die from pathology in the GI tract. Patchy hyperplasia of Kit-positive cells is evident within the myenteric plexus of the entire GI tract. Neoplastic lesions indistinguishable from human GISTs were observed in the cecum of the mutant mice with high penetrance. In addition, mast cell numbers in the dorsal skin were increased. Therefore KitV558Delta/+ mice reproduce human familial GISTs, and they may be used as a model for the study of the role and mechanisms of Kit in neoplasia. Importantly, these results demonstrate that constitutive Kit signaling is critical and sufficient for induction of GIST and hyperplasia of interstitial cells of Cajal.

Background: Adverse reactions after ingestion of alcoholic beverages are common. Metabolic differences in individuals and also the histamine content in alcoholic beverages have been implicated. By contrast pure ethanol has rarely been reported as a cause of hypersensitivity reactions and its mechanism has not been clarified yet., Objective: To determine whether ethanol itself accounts for alcohol hypersensitivity in patients with anaphylactic reactions after alcohol intake. In search of possible pathomechanisms all patients were analysed by skin prick testing and sulfidoleukotriene production of peripheral leucocytes using ethanol and its metabolites., Methods: Double-blind, placebo-controlled food challenges with a cumulated amount of 30 mL ethanol were performed in 12 adult patients with a positive history of adverse reactions after consumption of different alcoholic beverages. Skin prick tests and measurement of sulfidoleukotriene production were performed using different concentrations of ethanol and acetaldehyde from 50 to 1000 mm., Results: Oral challenges with pure ethanol were positive in six out of eleven patients. All challenge-positive patients, but also four out of five challenge-negative patients, showed an increased sulfidoleukotriene production in-vitro compared with healthy controls. Skin prick tests using alcoholic beverages, ethanol, acetaldehyde and acetic acid were negative in all patients (12/12)., Conclusion: Our study shows that ethanol itself is a common causative factor in hypersensitivity reactions to alcoholic beverages. These reactions occur dose-dependent and a non-IgE-mediated pathomechanism is likely, because skin prick tests were negative in all cases. Increased sulfidoleukotriene production was determined in some patients, but is no reliable predictor. Therefore oral provocation tests remain indispensable in making the diagnosis of ethanol hypersensitivity.

Many atopic dermatitis (AD) patients follow elimination diets for long periods of time because of true or--even more often--suspected food allergies or hypersensitivities. In the present study we investigated the nutritional status of adult AD patients by evaluating food intake protocols followed by measuring different serum parameters. Evaluation of the food intake protocols over 3 days (n = 47) showed that the total calorie intake met recommended intake in 12 patients (group A), ranged between 75-100% of the requirements in 23 patients (group B) and was 50-75% in 12 patients (group C). The relative intake of different nutritional factors approached normal limits in group A and B; the intake of several of these factors was significantly decreased in group C. Various serum parameters in group C patients were different from those found in patients suffering from other chronic skin diseases. Taken together, our results show that a subgroup of AD patients have dietary habits which result in significant nutritional deficiency which are important for homeostasis. This indicates the necessity of professional guidance in these patients in order to only eliminate foods which have been proven to elicit adverse reactions by oral provocation tests.

Patients with atopic dermatitis frequently blame food for worsening their eczema. Recently, a sugar-free diet has become very popular among German patients. In order to investigate whether sucrose is an aggravating factor in atopic dermatitis, we conducted a prospective study in 30 patients. Double-blind, placebo-controlled food challenges with sugar were performed during a sugar-free diet period, which was started one week prior to the first challenge. Study parameters included the SCORAD index and eosinophilic cationic protein levels, which were determined before diet, on food challenges and one day after. In 29 evaluable patients we observed no significant difference in study parameters after the verum challenge compared to the placebo challenge. From these results, we conclude that sugar is not an aggravating factor in atopic dermatitis, which contradicts the view of many patients and underlines the importance of evidence-based medicine in the guidance of patients.

Recently, we identified a subgroup of patients with atopic dermatitis (AD) with a clinical relevant food intolerance proven by double blind placebo controlled challenge. In search of possible pathomechanisms involved in this food intolerance, which leads to aggravation of the disease, the aim of the present study was to determine sulfidoleukotriene production in these patients using isolated leucocytes from the peripheral blood after stimulation with different food additives. Leukotriene production of peripheral leucocytes was detected by incubation of isolated cells with the food additives at different concentrations ranging from 0.2 to 200 microg/mL after pre-stimulation with IL-3. Ten non-atopic donors (A), nine AD patients of the diet responder group with negative oral provocation test against food additives (B) and nine patients of the responder group with positive reactions after the oral provocation test (C) were investigated. In the non-atopic group (A), no increased sulfidoleukotriene (sLT) release was observed for all food additives tested. In group B, increased sLT production was determined using tartrazine in one patient (1/9) and using nitrite in two patients (2/9), whereas sLT production remained below the cut-off range in all patients of group B (9/9) using benzoate, metabisulfite and salicylate. By contrast, in group C increased sLT production was observed with food colour mix in 1/9, with tartrazine in 3/9, with benzoate in 4/9, with nitrite in 5/9, with salicylate in 2/9 and with metabisulfite in 1/9. However, no increased sLT concentration was determined in the presence of the tested food additives in two patients of group C. Increased sLT production by peripheral leucocytes in the presence of single food additives was observed in the majority of patients with a proven food intolerance towards food additives proven by double-blind-placebo-controlled challenges. These food additives were particulary tartrazine, benzoate and nitrite. These findings indicate that single food additives as aggravating factors in AD patients may trigger the disease through increased sLT production as a pathophysiological mechanism.

Background: Adverse reactions to food play an important role in the pathogenesis of atopic dermatitis (AD). In infancy and childhood, food allergies are observed in up to 30%, whereas nonallergic hypersensitivity reactions (pseudoallergic reactions) towards food additives have been reported to occur between 2 and 7%. By contrast, sensitizations towards food allergens are rarely of clinical relevance in adults and little data is available on nonallergic hypersensitivity reactions. To date the role of pseudoallergic reactions as an aggravating factor in AD of adult patients remains controversial. However, many adult patients report on food-related aggravation of the disease and nonallergic hypersensitivity reactions have been incriminated repeatedly., Objective: To elucidate the relevance of food additives in adult patients suffering from AD., Methods: Fifty patients were monitored over 4 weeks under regular diet followed by 6 weeks of a diet omitting known pseudoallergens. Skin status of patients was assessed every 2 weeks by a standardized scoring, and serum eosinophilic cationic protein (ECP) was determined before and after diet., Results: Nine of fifty patients dropped out, 26 showed a significant improvement of the Costa-score by 57%. In 23/26 patients a corresponding reduction of serum ECP level by 52% on average was determined. Responder patients (24/26) were orally challenged with food rich in pseudoallergens followed by double-blind exposure to food additives (n = 15). A worsening of the eczema was seen in 19/24 patients after intake of pseudoallergen-rich food and in 6/15 patients after exposure to food additives., Conclusion: These results indicate that a subgroup of adult patients with AD clinically improve on low-pseudoallergen diet but only a small subgroup respond to oral provocation with food additives.

Background: IgE-independent (pseudoallergic) reactions to food and food ingredients are common in a subgroup of adult patients with chronic urticaria, who have daily spontaneous occurrence of wheals. However, for children with chronic urticaria (duration longer than 6 weeks, no physical influence), no data on the importance of pseudoallergen-induced chronic urticaria are available. Therefore, we investigated the role of nonallergic hypersensitivity to food in all children seen with chronic continuous urticaria in our two clinics over the last 2 years (n = 16)., Methods: All patients were given a low-pseudoallergen diet for 3 weeks followed by provocation with food rich in pseudoallergens. To identify the main eliciting agents, a subgroup of responders was exposed to food additives by double-blind, placebo-controlled food challenges., Results: Pseudoallergen-induced urticaria was diagnosed in 12 cases (75%). Reactions occurred mainly to coloring agents and preservatives, but also to monosodium glutamate and a sweetener (saccharin/cyclamate)., Conclusions: These results confirm that nonallergic hypersensitivity reactions play a role in children with chronic urticaria, although the latter disease is rare at that age. In children, food additives, especially coloring agents and preservatives, appear to play a more important role in eliciting nonallergic hypersensitivity reactions than in adult patients, where naturally occurring pseudoallergens in fruits and vegetables are mainly responsible.