PI3-Kinase Regulates Eosinophil and Neutrophil Degranulation in Patients with Allergic Rhinitis and Allergic Asthma Irrespective of Allergen Challenge Model

The PI3K pathway plays a major role in many vital cell processes. Our primary aim was to investigate signalling through PI3K for in vitro degranulation from allergen-primed eosinophils and neutrophils in allergic rhinitis and allergic asthma after seasonal and experimental allergen challenge. Nine patients with allergic rhinitis, eight with allergic asthma and four controls were studied during birch pollen season and after nasal and bronchial allergen challenge. Primed blood eosinophils and neutrophils were stimulated for in vitro degranulation with C3b-coated Sephadex particles, after prior incubation with Wortmannin, a PI3K inhibitor. The released amounts of eosinophil cationic protein (ECP), eosinophil peroxidase (EPO) and myeloperoxidase (MPO) were measured by radioimmunoassay. Wortmannin (10⁻⁶ to 10⁻⁹ M) inhibited ECP, EPO and MPO release in a dose-dependent manner in allergic rhinitis and allergic asthma in all three allergen challenge models. Inhibition of ECP release tended to be lower in the asthmatics in all allergen challenge models, statistically significant compared to the controls during season for 10⁻⁸ M Wortmannin (p = 0.01). A clear propensity towards less inhibition in the rhinitic patients was seen after nasal and bronchial challenge compared to seasonal exposure, significant for ECP (10⁻⁸ M Wortmannin; p = 0.034 and 0.002, respectively). Signalling through PI3K is clearly involved in ECP, EPO and MPO release in allergic rhinitis and allergic asthma irrespective of allergen challenge model. Allergic asthma demonstrated less inhibition of ECP release via PI3K during pollen season, indicating that other pathways play a greater role in eosinophil degranulation in allergic asthma than allergic rhinitis.     

The Fanconi anemia DNA damage repair pathway in the spotlight for germline predisposition to colorectal cancer

Colorectal cancer (CRC) is one of the most common neoplasms in the world. Fanconi anemia (FA) is a very rare genetic disease causing bone marrow failure, congenital growth abnormalities and cancer predisposition. The comprehensive FA DNA damage repair pathway requires the collaboration of 53 proteins and it is necessary to restore genome integrity by efficiently repairing damaged DNA. A link between FA genes in breast and ovarian cancer germline predisposition has been previously suggested. We selected 74 CRC patients from 40 unrelated Spanish families with strong CRC aggregation compatible with an autosomal dominant pattern of inheritance and without mutations in known hereditary CRC genes and performed germline DNA whole-exome sequencing with the aim of finding new candidate germline predisposition variants. After sequencing and data analysis, variant prioritization selected only those very rare alterations, producing a putative loss of function and located in genes with a role compatible with cancer. We detected an enrichment for variants in FA DNA damage repair pathway genes in our familial CRC cohort as 6 families carried heterozygous, rare, potentially pathogenic variants located in BRCA2/FANCD1, BRIP1/FANCJ, FANCC, FANCE and REV3L/POLZ. In conclusion, the FA DNA damage repair pathway may play an important role in the inherited predisposition to CRC.     

Alternatively Activated Macrophages and Collagen Remodeling Characterize the Postpartum Involuting Mammary Gland across Species

Recent pregnancy correlates with decreased survival for breast cancer patients compared with non–pregnancy-associated breast cancer. We hypothesize that postpartum mammary involution induces metastasis through wound-healing programs known to promote cancer. It is unknown whether alternatively activated M2 macrophages, immune cells important in wound-healing and experimental tumorigenesis that also predict poor prognosis for breast cancer patients, are recruited to the normal involuting gland. Macrophage markers CD68, CSF-1R, and F4/80 were examined across the pregnancy and involution cycle in rodent and human mammary tissues. Quantitative immunohistochemistry revealed up to an eightfold increase in macrophage number during involution, which returned to nulliparous levels with full regression. The involution macrophages exhibit an M2 phenotype as determined by high arginase-1 and low inducible nitric oxide synthase staining in rodent tissue, and by mannose receptor expression in human breast tissue. M2 cytokines IL-4 and IL-13 also peaked during involution. Extracellular matrix (ECM) isolated from involuting rat mammary glands was chemotactic for macrophages compared with nulliparous mammary ECM. Fibrillar collagen levels and proteolysis increased dramatically during involution, and denatured collagen I acted as a strong chemoattractant for macrophages in cell culture, suggesting proteolyzed fibrillar collagen as a candidate ECM mediator of macrophage recruitment. M2 macrophages, IL-4, IL-13, fibrillar collagen accumulation, and proteolysis of collagen are all components of tumor promotional microenvironments, and thus may mediate promotion of breast cancers arising in the postpartum setting.Although it is recognized that full-term pregnancy at an early age reduces the lifetime risk of developing breast cancer, women of all ages have a transient increase in breast cancer risk with a recent pregnancy.^1–4 Breast cancers diagnosed up to five years out from a completed pregnancy have been referred to as pregnancy-associated or PABC.^5,6 Several studies have shown that PABC frequently metastasizes, resulting in poor prognosis for the patient.^6–8 Epidemiological data identify women whose breast cancer is diagnosed postpartum, rather than during pregnancy, as having the worst outcomes.^7–14 Further, when breast cancer patients were matched for known prognostic indicators, the postpartum window proved to be an independent factor for metastasis, whereas a diagnosis during pregnancy did not.^11,14,15 We have proposed the involution-hypothesis to account for the high metastatic occurrence of PABC diagnosed during the postpartum window.^5,16–18 Specifically, we predict that the involuting microenvironment, with its similarities to wound and cancer environments, supports dissemination of pre-existing but previously quiescent tumor cells. In support of this hypothesis, we have shown that extracellular matrix (ECM) proteins isolated from the involution microenvironment have wound healing characteristics and are promotional for breast tumor cell metastasis.¹⁶ We extend our studies to address whether alternatively activated M2 macrophages, an immune cell type implicated in wound healing and breast cancer metastasis, participate in postpartum gland regression.Postpartum mammary gland involution is a necessary physiological process required to return the lactation competent gland to a nonlactating state poised to respond to a subsequent round of pregnancy. In rodent models, weaning-induced milk stasis triggers cessation of milk secretion and the milk-producing secretory alveoli that developed during pregnancy are rapidly resorbed. In rodent models, it has been shown that 50% to 80% of the secretory mammary epithelium is eliminated by apoptosis within the first week of involution.^19,20 This dramatic tissue remodeling requires two major processes: apoptotic death of the mammary epithelial cells and stromal activation.²¹ Accumulating evidence in rodents indicates that tissue-remodeling programs similar to wound healing are used to remodel the lactating gland to its postpartum state. Similarities include elevated levels of the immunomodulators transforming growth factor (TGF)-β1 and 3, increases in matrix metalloproteinase (MMP)-2, −3, and −9, deposition of fibrillar collagens, presence of bioactive proteolytic fragments of the ECM proteins fibronectin and laminin, as well as gene expression profiles consistent with activation of innate and adaptive immunity.^22–25Macrophages are a dynamic population of immune cells recognized for their contribution to wound resolution through phagocytosis and for their role in antigen presentation. The detailed functions of macrophages are extensive, as their precursor cells can respond to a variety of physiological situations and mature along a spectrum of phenotypes. The M1/M2 macrophage nomenclature system described by Mantovani and colleagues²⁶ primarily divides cells based on production of Th1 versus Th2 cytokines. According to this classification, the M1-type, or classically activated macrophages, mature in response to signaling molecules elicited by intracellular pathogens including lipopolysaccharide, interferon-γ and -β, and TNF-α. These M1 macrophages respond in kind by producing Th1 cytokines including interleukin (IL)-12 and IL-23 that subsequently promote activation of cytotoxic T-lymphocytes. M1 macrophages are involved in antigen presentation, immune surveillance, and killing of cells with foreign antigens, including tumor cells.^26–28On the other end of the spectrum, M2 macrophages are alternatively activated by Th2 cytokines such as IL-4, IL-13, and the immunosuppressive IL-10.²⁶ M2-type macrophages in turn produce Th2 cytokines. M2 macrophages participate in wound healing and tissue repair through activities including phagocytic debris clearance, release of TGF-β and fibronectin, and release of PDGF and VEGF,^27,29 which promote angiogenesis. Recently, it has become apparent that macrophages are functionally plastic, in that they respond to their environment with heterogeneous and transient phenotypes that are not distinctly divided into the M1/M2 categories.³⁰Several recent reviews highlight shared characteristics of M2 macrophages and tumor-associated macrophages (TAMs).^26,31 TAMs are important in the progression of mammary cancer in several rodent models.^32,33 The observed similarities between TAMs and M2 macrophages suggest that M2 macrophages may also have tumor promotional capabilities.³⁴ Further, clinical data reveals that macrophage chemotactic factors, macrophage growth factors, and macrophage infiltration all correlate with negative outcomes for breast cancer patients.^35–40 TAMs and M2 macrophages are thus implicated as key mediators of breast cancer progression.Based on evidence that macrophages promote breast cancer and that the wound-healing microenvironment of the involuting rodent mammary gland is tumor promotional, we evaluated whether macrophages characterize the postpartum involuting mammary gland. Here we demonstrate an influx of macrophages during postlactational involution, identified as consisting primarily of M2 macrophages in rodents and humans. Further, we provide a plausible mechanism for macrophage recruitment via their attraction to the ECM protein collagen I. These observations are consistent with our previous data demonstrating that the involuting rat mammary gland is characterized by a wound healing microenvironment and extends these findings to human breast tissues. These data further support the hypothesis that postpartum involution promotes tumor progression and accounts for the poor prognosis of PABC.     

Diploid-dominant life cycles characterize the early evolution of Fungi

Most of the described species in kingdom Fungi are contained in two phyla, the Ascomycota and the Basidiomycota (subkingdom Dikarya). As a result, our understanding of the biology of the kingdom is heavily influenced by traits observed in Dikarya, such as aerial spore dispersal and life cycles dominated by mitosis of haploid nuclei. We now appreciate that Fungi comprises numerous phylum-level lineages in addition to those of Dikarya, but the phylogeny and genetic characteristics of most of these lineages are poorly understood due to limited genome sampling. Here, we addressed major evolutionary trends in the non-Dikarya fungi by phylogenomic analysis of 69 newly generated draft genome sequences of the zoosporic (flagellated) lineages of true fungi. Our phylogeny indicated five lineages of zoosporic fungi and placed Blastocladiomycota, which has an alternation of haploid and diploid generations, as branching closer to the Dikarya than to the Chytridiomyceta. Our estimates of heterozygosity based on genome sequence data indicate that the zoosporic lineages plus the Zoopagomycota are frequently characterized by diploid-dominant life cycles. We mapped additional traits, such as ancestral cell-cycle regulators, cell-membrane– and cell-wall–associated genes, and the use of the amino acid selenocysteine on the phylogeny and found that these ancestral traits that are shared with Metazoa have been subject to extensive parallel loss across zoosporic lineages. Together, our results indicate a gradual transition in the genetics and cell biology of fungi from their ancestor and caution against assuming that traits measured in Dikarya are typical of other fungal lineages.

Fungi and Metazoa evolved from a common protist-like ancestor, yet the two kingdoms have diverged in ways that make their kinship as Opisthokonts barely recognizable. Fungi grow on or within their food and feed by external digestion (osmotrophy), while animals mostly eat things smaller than themselves via ingestion. This difference is the basis for massive changes in morphology, including loss of motility during feeding and polarized cell growth in fungi (1, 2). The two kingdoms are also considered intrinsically different in life cycles, because fungi are characterized as being haplontic (haploid-dominant life cycle) while animals are diplontic (diploid-dominant). However, this textbook difference is inaccurate in two ways. First, the subkingdom Dikarya, with the majority of fungal species diversity, comprises lineages that spend some or most of their life cycles in a dikaryotic phase wherein two haploid nuclei undergo conjugate division, a cell type genetically analogous to a diploid (3). Further, life cycles have not been carefully investigated in most early-diverging fungal lineages (EDF), which include many phyla outside of Dikarya (e.g., non-Dikarya fungi). EDF have retained ancestral traits also retained in Metazoa, such as flagellation, actin structures used for crawling, presence of cholesterol in cell membranes, vitamin dependencies, and cell-cycle genes (4–8). However, life-cycle transitions between the Opisthokont ancestor and the extant Fungi are shrouded due to a lack of information on the genetic characteristics of EDF and the undersampling of their genomic diversity (9–11). The goal of this paper is to provide a robust and comprehensive phylogeny of the Fungi, emphasizing zoosporic taxa, to reassess the evolution of life-cycle and cellular characters during early fungal diversification using genomic data.Although fungi are often considered to have haploid-dominant life cycles, there are many variations observed (Fig. 1). In a haplontic life cycle, mitosis is restricted to the haploid phase, and meiosis ensues immediately following sex and nuclear fusion (Fig. 1A). In contrast, in the diplontic life cycle that generally characterizes Metazoa, mitosis is restricted to diploid cells (Fig. 1B). The alternation between haploid and diploid mitotic cycles, which generally characterizes plants, is documented, albeit rarely, in fungi, such as baker’s yeast and the water mold Allomyces (Fig. 1C). Despite this general avoidance of diploid mitosis in fungi, many Dikarya show a distinctive dikaryotic life cycle wherein, following mating, haploid nuclei of the two partners remain paired and undergo synchronous mitoses (Fig. 1D). This life cycle is analogous to diploidy with respect to genetic dominance (12) and would provide some of the proposed advantages of diploidy, such as buffering against somatic mutation (13). Overall, although we appreciate that fungal life cycles have great potential to vary, we have a poor understanding of life cycles of the EDF which represent the majority of the phylogenetic diversity of Fungi.Open in a separate windowFig. 1.Illustrated life cycles observed in fungi. (A) In haplontic life cycles mitosis is limited to the haploid phase, with plasmogamy of gametes followed by meiosis. (B) In diplontic life cycles, mitosis only occurs in the diploid phase with haploid cells only functioning as gametes. (C) Life cycles may alternative between haploid and diploid mitotic phases and may show morphological differences between ploidies as in Allomyces. (D) The dikaryotic life cycle is an alternative to alternation of haploid and diploid generations which lacks diploid mitosis and instead has a phase with two nuclear genotypes undergoing synchronous division.We consider EDF to comprise 11 phyla, including 8 zoosporic phyla that reproduce with swimming spores and form a contentious paraphyletic grade along the backbone of the fungal tree (9, 10, 14–16). The deeply diverging phyla, Rozellomycota/Cryptomycota and Aphelidiomycota, are endoparasites that have the ability to phagocytize, which enables them to ingest host cytoplasm, a trait presumably retained from the most recent common ancestor (MRCA) of Opisthokonta (17, 18). The remaining free-living zoosporic phyla have microscopic vegetative thalli that may be unicellular or mycelial (SI Appendix, Fig. S1), and the greatest species diversity is found in the Chytridiomycota, which has an estimated 14 orders (9). Chytridiomycota is united with the phyla Monoblepharidomycota + Neocallimastigomycota in subkingdom Chytridiomyceta, though the branching order of the three phyla is uncertain (14, 15, 19).Blastocladiomycota is an enigmatic group with a life cycle alternating between morphologically distinctive haploid and diploid thalli (Fig. 1C) (20, 21). Members include the water mold, Allomyces, that has been used as a model system for genetics and physiology (22) and a genus of obligate fatal parasites, Coelomomyces, that has a haploid phase in copepods and a diploid phase in mosquitoes (23). The precise phylogenetic placement of the Blastocladiomycota has been controversial (10, 15, 19, 24), with nearly equal support for the Blastocladiomycota diverging before the Chytridiomyceta or after the Chytridiomyceta. Several traits of Blastocladiomycota ally them with the terrestrial fungi (here defined as the nonzoosporic phyla Mucoromycota and Zoopagomycota and subkingdom Dikarya): closed mitosis, the presence of a Spitzenkörper, beta 1,3 glucans in the cell wall, and true mycelial growth in some members (22, 25). The detection of mating types in Coelomomyces (26), which have only been otherwise documented in terrestrial fungi, may be indicative that Blastocladiomycota is more closely related to Dikarya than Chytridiomyceta.Mating and sexuality are poorly described in zoosporic fungi beyond the well-characterized water mold model Allomyces. According to mycological textbooks, life cycles of Chytridiomycota are characterized as being haplontic with zygotic meiosis (27–29), but the majority of assumptions of meiotic stages are unconfirmed by cytology. Moreover, the requisite genetic studies using molecular markers to confirm ploidy cycling have not been accomplished for these presumably sexual species. Importantly, the best-studied chytrid fungus, Batrachochytrium dendrobatidis, has a life cycle that appears to be dominated by asexually reproducing diploid, or aneuploid, thalli (30). More recently, additional studies have indicated that non-Dikarya phyla have heterozygosity indicative of diploidy or higher ploidy (31–34), suggesting that the assumption of haplontic life cycles for the Chytridiomyceta and other EDF may be false.Current sequencing technologies now create the potential for leveraging genomic sequencing to broadly sample fungal genomes for estimating ploidy and other cellular traits in a robust phylogenomic framework. Here, we sampled 69 zoosporic fungal genomes using both culture and single-cell approaches. Our genome analyses provide a strongly supported phylogeny for understanding taxonomy and the evolution of ploidy and other traits which had previously been held to be distinctive between Fungi and Metazoa. These data bolster the growing picture that many traits including motility, feeding modes, and life cycles changed gradually during the early diversification of fungi. The high levels of heterozygosity estimated from genomes analyzed in this study reveal that somatic diploidy is much more common in Fungi than previously appreciated.     

Evaluation of several creatinine methods in search of a suitable secondary reference method: report from the subcommittee on reference method for creatinine, Nordic Society for Clinical Chemistry

Several methods for creatinine have been examined by the subcommittee on reference method for creatinine. These include the use of picric acid to precipitate proteins followed by alkalization, or the use of alkaline picrate after protein precipitation and adsorption to Fuller's earth. The first approach was not found to be suitable as a reference method since it was susceptible to matrix effects, fluctuations in temperature and wavelengths. The second approach was less sensitive to these effects but showed variations with different batches of Fuller's earth. The method using high performance liquid chromatography (HPLC) had poor precision, probably because endogenous compounds co-eluted either with creatinine or with the internal standard. The enzymatic method based on the determination of hydrogen peroxide had good accuracy and precision. However, the main disadvantage is that the method is sold as a kit and detailed specifications concerning all the ingredients are not available to permit independent studies using the basic components.     

Changes in intensity of physical exercise as predictors of depressive symptoms among older adults: an eight-year follow-up

BACKGROUND: Epidemiological research suggests that regular physical activity may be associated with reduced depressive symptoms. The present study examines the predictive value of physical exercise in relation to depressive symptoms among samples of adults aged 65+ during an 8-year period. METHODS: The subjects (N = 663) who participated both at the baseline (1988) and the follow-up (1996) interviews were selected for the analyses. The dependent variable depressive symptoms was assessed by the Finnish modified version of Beck's 13-item depression scale. The independent variable was the intensity of physical exercise. RESULTS: The intensity of physical exercise decreased among the older men and women. Those who had reduced their intensity of physical exercise during the 8 years reported more depressive symptoms at the follow-up than those who had remained active or increased their physical activity. Depressive symptoms were predicted by the intensity of baseline physical exercise, earlier depressive symptoms, older age, gender, having three or more chronic somatic conditions, and difficulties in performing ADL activities. CONCLUSIONS: Age-related decrease in the intensity of physical exercise increases the risk of depressive symptoms among older adults. This calls for effective measures in maintaining and supporting an adequate level of physical exercise among the aging population.