Apolipoprotein E controls Dectin-1-dependent development of monocyte-derived alveolar macrophages upon pulmonary ß-glucan-induced inflammatory adaptation.

The lung is constantly exposed to the outside world and optimal adaptation of immune responses is crucial for efficient pathogen clearance. However, mechanisms that lead to lung-associated macrophages' functional and developmental adaptation remain elusive. To reveal such mechanisms, we developed a reductionist model of environmental intranasal ß-glucan exposure, allowing for the detailed interrogation of molecular mechanisms of pulmonary macrophage adaptation. Employing single-cell transcriptomics, high-dimensional imaging and flow cytometric characterization paired with in vivo and ex vivo challenge models, we reveal that pulmonary low-grade inflammation results in the development of apolipoprotein E (ApoE)-dependent monocyte-derived alveolar macrophages (ApoE+CD11b+ AMs). ApoE+CD11b+ AMs expressed high levels of CD11b, ApoE, Gpnmb and Ccl6, were glycolytic, highly phagocytic and produced large amounts of interleukin-6 upon restimulation. Functional differences were cell intrinsic, and myeloid cell-specific ApoE ablation inhibited Ly6c+ monocyte to ApoE+CD11b+ AM differentiation dependent on macrophage colony-stimulating factor secretion, promoting ApoE+CD11b+ AM cell death and thus impeding ApoE+CD11b+ AM maintenance. In vivo, ß-glucan-elicited ApoE+CD11b+ AMs limited the bacterial burden of Legionella pneumophilia after infection and improved the disease outcome in vivo and ex vivo in a murine lung fibrosis model. Collectively these data identify ApoE+CD11b+ AMs generated upon environmental cues, under the control of ApoE signaling, as an essential determinant for lung adaptation enhancing tissue resilience.

Painful skin lesions and squamous cell carcinoma predict overall mortality risk in organ transplant recipients: a cohort study.

BACKGROUND: Organ transplant recipients (OTRs) have a highly increased risk of cutaneous squamous cell carcinomas (SCCs). Sensation of pain in cutaneous tumours is a powerful patient-reported warning signal for invasive SCCs in OTRs. OBJECTIVES: To investigate the impact of painful vs. painless skin lesions and SCC vs. other skin lesions on the overall mortality risk in OTRs. METHODS: We followed 410 OTRs from 10 different centres across Europe and North America between 2008 and 2015. These patients had been enrolled in an earlier study to define clinically meaningful patient-reported warning signals predicting the presence of SCC, and had been included if they had a lesion requiring histological diagnosis. Cumulative incidences of overall mortality were calculated using Kaplan-Meier survival analysis, and risk factors were analysed with Cox proportional hazard analysis. RESULTS: There was an increased overall mortality risk in OTRs who reported painful vs. painless skin lesions, with a hazard ratio (HR) of 1·6 [95% confidence interval (CI) 0·97-2·7], adjusted for age, sex and other relevant factors. There was also an increased overall mortality risk in OTRs diagnosed with SCC compared with other skin lesions, with an adjusted HR of 1·7 (95% CI 1·0-2·8). Mortality due to internal malignancies and systemic infections appeared to prevail in OTRs with SCC. CONCLUSIONS: We suggest that OTRs have an increased overall mortality risk if they develop painful skin lesions or are diagnosed with cutaneous SCC.

Epidermal barrier abnormalities in exfoliative ichthyosis with a novel homozygous loss-of-function mutation in CSTA.

Autosomal recessive exfoliative ichthyosis (AREI) results from mutations in CSTA, encoding cysteine protease inhibitor A (cystatin A). We present a 25-year-old man from Iran with consanguineous parents, who presented with congenital erythroderma, hyperhidrosis and diffuse hyperkeratosis with coarse palmoplantar peeling of the skin, aggravated by exposure to water and by occlusion. Candidate gene analysis revealed a previously unknown homozygous loss-of-function mutation c.172C>T (p.Arg58Ter) in CSTA, and immunostaining showed absence of epidermal cystatin A, confirming the diagnosis of AREI. Ultrastructural analysis by transmission electron microscopy showed normal degradation of corneodesmosomes, mild intercellular oedema in the spinous layer but not in the basal layer, normal-appearing desmosomes, and prominent keratin filaments within basal keratinocytes. Thickness of cornified envelopes was reduced, lamellar lipid bilayers were disturbed, lamellar body secretion occurred prematurely and processing of secreted lamellar body contents was delayed. These barrier abnormalities were reminiscent of (albeit less severe than in) Netherton syndrome, which results from a deficiency of the serine protease inhibitor LEKTI. This work describes ultrastructural findings with evidence of epidermal barrier abnormalities in AREI.

A cross-species approach to identify transcriptional regulators exemplified for Dnajc22 and Hnf4a.

There is an enormous need to make better use of the ever increasing wealth of publicly available genomic information and to utilize the tremendous progress in computational approaches in the life sciences. Transcriptional regulation of protein-coding genes is a major mechanism of controlling cellular functions. However, the myriad of transcription factors potentially controlling transcription of any given gene makes it often difficult to quickly identify the biological relevant transcription factors. Here, we report on the identification of Hnf4a as a major transcription factor of the so far unstudied DnaJ heat shock protein family (Hsp40) member C22 (Dnajc22). We propose an approach utilizing recent advances in computational biology and the wealth of publicly available genomic information guiding the identification of potential transcription factor candidates together with wet-lab experiments validating computational models. More specifically, the combined use of co-expression analyses based on self-organizing maps with sequence-based transcription factor binding prediction led to the identification of Hnf4a as the potential transcriptional regulator for Dnajc22 which was further corroborated using publicly available datasets on Hnf4a. Following this procedure, we determined its functional binding site in the murine Dnajc22 locus using ChIP-qPCR and luciferase assays and verified this regulatory loop in fruitfly, zebrafish, and humans.

Avarol-induced DNA strand breakage in vitro and in Friend erythroleukemia cells.

The hydroquinone-containing cytostatic compound avarol inhibits predominantly growth of those cell lines which have a low level of superoxide dismutase. The substrate of this enzyme, the superoxide anion, was found to be formed during the in vitro oxidation reaction of avarol to its semiquinone radical in the presence of oxygen. Under the same incubation conditions plasmid DNA (pBR322) was converted from the fully supercoiled circular form mainly to the nicked circular form, indicating that the compound causes primarily single-strand breaks. Using Friend erythroleukemia cells (FLC) it was found that avarol induces a dose-dependent DNA damage; the maximum number of DNA strand breaks was observed at 5 h after addition of the compound to the cells. Removal of avarol resulted in a rapid DNA rejoining with biphasic repair kinetics [first half-time, 8 min (90% of the breaks) and a second half-time, 40 min (10% of the breaks)]. When the degree of avarol-induced DNA damage in FLC was compared with the drug-caused inhibition of cell growth a close correlation was established. Avarol displayed no effect on dimethyl sulfoxide-induced erythrodifferentiation of FLC as determined by the benzidine reaction and by dot blot hybridization experiments. From incubation studies of FLC with [3H]avarol no hint was obtained for the formation of an adduct between DNA and the compound. The subcellular distribution of [3H]avarol was studied in liver cells after i.v. application of the compound. The predominant amount of the compound was present in the cytosolic fraction; little avarol was associated with plasma membranes, nuclei, and mitochondria. Using (a) oxidative phosphorylation and (b) oxygen uptake as parameters for mitochondria function, no effect of the compound on the activity of this organelle was determined. These results suggest that avarol forms superoxide anions (and in consequence possibly also hydroxyl radicals) especially in those cells which have low levels of superoxide dismutase. Moreover, evidence is provided that the active oxygen species cause DNA damage resulting in the observed cytotoxic effect.

Extreme Thouless effect in a minimal model of dynamic social networks.

In common descriptions of phase transitions, first-order transitions are characterized by discontinuous jumps in the order parameter and normal fluctuations, while second-order transitions are associated with no jumps and anomalous fluctuations. Outside this paradigm are systems exhibiting "mixed-order" transitions displaying a mixture of these characteristics. When the jump is maximal and the fluctuations range over the entire range of allowed values, the behavior has been coined an "extreme Thouless effect." Here we report findings of such a phenomenon in the context of dynamic, social networks. Defined by minimal rules of evolution, it describes a population of extreme introverts and extroverts, who prefer to have contacts with, respectively, no one or everyone. From the dynamics, we derive an exact distribution of microstates in the stationary state. With only two control parameters, N(I,E) (the number of each subgroup), we study collective variables of interest, e.g., X, the total number of I-E links, and the degree distributions. Using simulations and mean-field theory, we provide evidence that this system displays an extreme Thouless effect. Specifically, the fraction X/(N(I)N(E)) jumps from 0 to 1 (in the thermodynamic limit) when N(I) crosses N(E), while all values appear with equal probability at N(I)=N(E).

Kit transduced signals counteract erythroid maturation by MAPK-dependent modulation of erythropoietin signaling and apoptosis induction in mouse fetal liver.

Signaling by the stem cell factor receptor Kit in hematopoietic stem and progenitor cells is functionally associated with the regulation of cellular proliferation, differentiation and survival. Expression of the receptor is downregulated upon terminal differentiation in most lineages, including red blood cell terminal maturation, suggesting that omission of Kit transduced signals is a prerequisite for the differentiation process to occur. However, the molecular mechanisms by which Kit signaling preserves the undifferentiated state of progenitor cells are not yet characterized in detail. In this study, we generated a mouse model for inducible expression of a Kit receptor carrying an activating mutation and studied its effects on fetal liver hematopoiesis. We found that sustained Kit signaling leads to expansion of erythroid precursors and interferes with terminal maturation beyond the erythroblast stage. Primary KIT(D816V) erythroblasts stimulated to differentiate fail to exit cell cycle and show elevated rates of apoptosis because of insufficient induction of survival factors. They further retain expression of progenitor cell associated factors c-Myc, c-Myb and GATA-2 and inefficiently upregulate erythroid transcription factors GATA-1, Klf1 and Tal1. In KIT(D816V) erythroblasts we found constitutive activation of the mitogen-activated protein kinase (MAPK) pathway, elevated expression of the src kinase family member Lyn and impaired Akt activation in response to erythropoietin. We demonstrate that the block in differentiation is partially rescued by MAPK inhibition, and completely rescued by the multikinase inhibitor Dasatinib. These results show that a crosstalk between Kit and erythropoietin receptor signaling cascades exists and that continuous Kit signaling, partly mediated by the MAPK pathway, interferes with this crosstalk.

Utilization of N-acetyl-L-tyrosine and glycyl-L-tyrosine during long-term parenteral nutrition in the growing rat.

Utilization of N-acetyl-L-tyrosine and glycyl-L-tyrosine as a source of tyrosine in infusion solutions was tested in rats receiving total parenteral nutrition for 4 wk. The four solutions tested were isonitrogenous and isocaloric. One of the solutions contained an adequate amount of L-phenylalanine; in the other three, two-thirds of the phenylalanine was replaced by a corresponding amount of either glycine, glycyl-L-tyrosine or N-acetyl-L-tyrosine. No differences in weight gain or N-balance could be detected as a result of administering either the solution with glycyl-L-tyrosine or with N-acetyl-L-tyrosine in place of the solution containing an adequate phenylalanine content. The solution in which two-thirds of the L-phenylalanine was replaced by glycine yielded only half of the weight gain and correspondingly reduced values for N-balance. Daily urinary excretion rates for N-acetyl-L-tyrosine and glycyl-L-tyrosine were 11% and 0.5%, respectively, of the infused amount. Plasma amino acid pattern was affected differently by the four solutions. The results indicate that both N-acetyl-L-tyrosine and glycyl-L-tyrosine are efficiently utilized by the rat during total parenteral nutrition.

Utilization of glutathione disulfide as cysteine source during long-term parenteral nutrition in the growing rat.

Utilization of intravenously administered glutathione disulfide was investigated during long-term parenteral nutrition in growing rats. In a series of cross-over studies, three solutions were tested against one another by recording weight gain, nitrogen balance, and plasma amino acid patterns. Solution 1 contained the required amount of methionine for rats, solution 2 had only one third of the required methionine, but was made isonitrogenous with glycine, whereas in solution 3, two thirds of the methionine was replaced by glutathione disulfide. Weight gain was about twice as high during infusion with either the required amount of methionine or the glutathione disulfide when compared with solution 2. Nitrogen retention was significantly higher during infusion with sufficient methionine or a corresponding amount of glutathione disulfide, when compared with the solution low in methionine. Plasma levels of cystine decreased significantly under the low methionine supply, but no difference was observed for the groups receiving sufficient methionine or the corresponding amount of glutathione disulfide. It is concluded that glutathione disulfide permits adequate cysteine supply in parenteral nutrition and may replace part of the methionine in the presence of an impaired conversion of methionine to cysteine.

Utilization of methionine and N-acetyl-L-cysteine during long-term parenteral nutrition in the growing rat.

Utilization of methionine and N-acetyl-L-cysteine as a source of cysteine was tested in growing rats receiving total parenteral nutrition for four weeks. The three solutions tested were isonitrogenous and isocaloric. One of the solutions contained an adequate amount of L-methionine, in the other two, two thirds of the L-methionine was substituted by a corresponding amount of either glycine or N-acetyl-L-cysteine. Weight gain and N-balance were similar under the infusion with either the adequate amount of L-methionine or the N-acetyl-L-cysteine substituted. The solution in which two thirds of the L-methionine was replaced by glycine yielded only half of the weight gain and correspondingly reduced values for N-balance. The daily urinary excretion rate for N-acetyl-L-cysteine was 4.6% of the infused amount. Urinary excretion rates of the other amino acids and the plasma amino acid pattern was affected differently by the three solutions. The results indicate that cysteine is more rapidly available from N-acetyl-L-cysteine than from L-methionine when administered intravenously.

Classification, clinical manifestations, and immunopathological mechanisms of the epithelial variant of paraneoplastic autoimmune multiorgan syndrome: a reappraisal of paraneoplastic pemphigus.

BACKGROUND: Recent studies suggest that paraneoplastic pemphigus (PNP) is a heterogeneous autoimmune syndrome involving several internal organs and that the pathophysiological mechanisms mediating cutaneous, mucosal, and internal lesions are not limited to autoantibodies targeting adhesion molecules. OBJECTIVE: To classify the diverse mucocutaneous and respiratory presentations of PNP and characterize the effectors of humoral and cellular autoimmunity mediating epithelial tissue damage. METHODS: We examined 3 patients manifesting the lichen planus pemphigoideslike subtype of PNP. A combination of standard immunohistochemical techniques, enzyme-linked immunosorbent assay with desmoglein (DSG) baculoproteins, and an immunoprecipitation assay were used to characterize effectors of humoral and cellular autoimmunity in patients with PNP and in neonatal wild-type and DSG3-knockout mice with PNP phenotype induced by passive transfer of patients' IgGs. RESULTS: In addition to the known "PNP antigenic complex," epithelial targets recognized by PNP antibodies included 240-, 150-, 130-, 95-, 80-, 70-, 66-, and 40/42-kd proteins but excluded DSG1 and DSG3. In addition to skin and the epithelium lining upper digestive and respiratory tract mucosa, deposits of autoantibodies were found in kidney, urinary bladder, and smooth as well as striated muscle. Autoreactive cellular cytotoxicity was mediated by CD8(+) cytotoxic T lymphocytes, CD56(+) natural killer cells, and CD68(+) monocytes/macrophages. Inducible nitric oxide synthase was visualized both in activated effectors of cellular cytotoxicity and their targets. Keratin 14-positive basal epithelial cells sloughed from the large airways and obstructed small airways. CONCLUSIONS: The paraneoplastic disease of epithelial adhesion known as PNP in fact represents only 1 manifestation of a heterogeneous autoimmune syndrome in which patients, in addition to small airway occlusion and deposition of autoantibodies in different organs, may display a spectrum of at least 5 different clinical and immunopathological mucocutaneous variants (ie, pemphiguslike, pemphigoidlike, erythema multiforme-like, graft-vs-host disease-like, and lichen planus-like). We suggest that the more encompassing term "paraneoplastic autoimmune multiorgan syndrome," or PAMS, be applied. The pathophysiological mechanisms of PAMS involve both humoral and cellular autoimmunity responses. Epithelial cell membrane antigens other than DSG1 or DSG3 are targeted by effectors of PAMS autoimmunity. Apoptosis of damaged basal cells mediates epithelial clefting, and respiratory failure results possibly from obstruction of small airways with sloughed epithelial cells.

The metabolic status of internal intensive care patients as indicated by 3-methylhistidine excretion and nitrogen balance.

The metabolic status of 15 intensive care patients receiving a standardized total parenteral nutrition regimen was followed up to 15 days immediately after admission by measuring 3-methylhistidine, total nitrogen, and creatinine excretion. The average 3-methylhistidine excretion was within the normal range during the first 3 days, rising on day 4 and reached a maximum of 70% above normal values on day 5. It declined to within normal range thereafter in most of the patients. Mean values for creatinine excretion remained relatively constant within the normal range throughout the study. During all days 3-methylhistidine was negatively correlated with N-balance. It is concluded that these patients had increasing catabolism with a maximum on day 5 and that the catabolic condition was associated with an increased muscle protein breakdown.

Rapid determination of vitamin A (retinol) and vitamin E (alpha-tocopherol) in human serum by isocratic adsorption HPLC.

The simultaneous determination of alpha-tocopherol and retinol in human serum is reported. The separation is carried out by means of isocratic HPLC on adsorption columns. UV-Detection is possible by using either one wavelength for both compounds (300 nm), or after a lambda change mode with typical wavelengths for alpha-tocopherol (292 nm) and retinol (325 nm). According to short retention times (10 min) and rapid extraction the method is useful for clinical research and allows about 50 analyses per day and operator. Blood from 176 human volunteers was collected and alpha-tocopherol and retinol levels in serum determined with this method. Statistical evaluation of different selected groups shows typical significant differences of alpha-tocopherol and retinol concentrations in smokers and oral contraceptive users.

PIP: An extraction and separation method for simultaneous vitamin A and E determination was developed to overcome problems of evaporation steps. The method did not involve an evaporation step and separation on adsorption columns by isocratic HPLC. It permits 50 analyses daily and operates with high sensitivity and reproducibility. Blood was collected from 115 healthy male and 63 healthy female volunteers ranging in age from 18-28 years. Retinol (25 mg) was dissolved in isopropanol and taken as stock solution. An aliquot of the stock solution was diluted and its absorbance measured at typical wave length against isopropanol and calculated. Alpha-Tocopherol was dissolved in isopropanol, an aliquot of this stock solution diluted its absorbance measured against isopropanol and calculated. Standard solutions were prepared by appropriate dilution of the measured stock solution. Retinol and a-tocopherol can be extracted in organic solvents after denaturation of the specific binding proteins by adding ethanol. A table shows that small amounts of serum and ethanol are sufficient to extract both vitamins quantitatively. Increasing a-tocopherol concentrations in ethanol can be extracted quantitatively in the hexane phase and, if added to hexane, the vitamin is not lost in the ethanol phase. The recovery study demonstrates different concentrations of the vitamin do not influence the quantitative extraction, and the recovery range indicates that the precision of the method is adequate within the linear range. The stock solution is stable more than 7 weeks when stored at -34 degrees Centigrade in glass vessels; the standard solutions should be prepared for each working day. The serum samples were stable more than 2 weeks when stored at -34 degrees Centigrade. The differences between the retinol values in selected groups are greater than the a-tocopherol differences. Retinol of females using oral contraceptives (OCs) was higher than of nonusers. This elevation was related to the estrogen content of the OCs. Cigarette smoking increased retinol levels slightly; in non-smokers the values were below the normal range. For OC users, the vitamin E levels did not show any significant differences. Cigarette smoking increased plasma vitamin A levels significantly but not vitamin E levels. The rapid simultaneous determination of retinol and a-tocopherol in plasma is useful in several clinical situations and in the nutritional assessment of normal subjects.

Uptake and metabolism of retinoic acid induces inhibition of cell growth: a study in a rat rhabdomyosarcoma cell line (BA-HAN-1C) using nonlinear theoretical models.

Retinoic acid causes a significant inhibition of cell growth of the tumor cell line BA-HAN-1C. This growth inhibition is the same whether the cells are treated with a pulse dose of retinoic acid (RA) or continuously expand to RA. The determination of RA and its degradation products within the culture medium and in the cells showed that after 24 hours 13-cis-RA was the major retinoid in all cells (96 ng/10(6) cells); all-trans-RA represented 56 ng/10(6) cells. After 48 hours 4-hydroxy-RA and a small amount of 5,6-epoxy-RA was found in the cells and also in the culture medium. 4-hydroxy-RA increased up to 96 hours, whereas 13-cis- and all-trans-RA were not detectable in the cells after 96 hours. We conclude that the BA-HAN-1C cells take up and metabolize RA. Nonlinear fit analysis of the time behavior of the RA concentration in medium demonstrates that the RA uptake unexpectedly follows a mono-exponential time function. Discussion of the experimental results in connection with a proper compartment model shows that uptake and metabolism of RA cannot be described really by a first order kinetics. The mathematical analysis leads to a more complicated kinetic model with certain restrictions for the corresponding rate constants.

Morphological instability in InAs/GaSb superlattices due to interfacial bonds.

Synchrotron x-ray diffraction is used to compare the misfit strain and composition in a self-organized nanowire array in an InAs/GaSb superlattice with InSb interfacial bonds to a planar InAs/GaSb superlattice with GaAs interfacial bonds. It is found that the morphological instability that occurs in the nanowire array results from the large misfit strain that the InSb interfacial bonds have in the nanowire array. Based on this result, we propose that tailoring the type of interfacial bonds during the epitaxial growth of III-V semiconductor films provides a novel approach for producing the technologically important morphological instability in anomalously thin layers.