Management of patients with severe asthma: results from a survey among allergists and clinical immunologists of the Central Italy Inter-Regional Section of SIAAIC.

BACKGROUND: Asthma, and severe asthma in particular, is often managed within a specialized field with allergists and clinical immunologists playing a leading role. In this respect, the National Scientific Society SIAAIC (Società Italiana di Allergologia, Asma ed Immunologia Clinica), structured in Regional and Inter-Regional sections, interviewed a large number of specialists involved in the management of this respiratory disease. METHODS: A survey entitled "Management of patients with asthma and severe asthma" based on 17 questions was conducted through the SIAAIC newsletter in 2019 thanks to the collaboration between GlaxoSmithKline S.p.A. and the Inter-Regional Section of SIAAIC of Central Italy. RESULTS: Fifty-nine allergists and clinical immunologists participated to the survey, and 40 of them completed the entire questionnaire. Almost all of the specialists (88%) reported that asthma control was achieved in above 50% of their patients, even if only one third (32%) actually used validated clinical tools such as asthma control test (ACT). Poor adherence to inhaled therapy was recognized as the main cause of asthma control failure by 60% of respondents, and 2-5 min on average is dedicated to the patient inhaler technique training by two-thirds of the experts (65%). Maintenance and as-needed therapy (SMART/MART) is considered an appropriate approach in only a minority of the patients (25%) by one half of the respondents (52%). A high number of exacerbations despite the maximum inhalation therapy were recognized as highly suspicious of severe asthma. Patients eligible for biological therapies are 3-5% of the patients, and almost all the responders (95%) agreed that patients affected by severe asthma need to be managed in specialized centers with dedicated settings. Biological drugs are generally prescribed after 3-6 months from the initial access to the center, and once started, the follow-up is initially programmed monthly, and then every 3-6 months after the first year of treatment (96% of responders). After phenotyping and severity assessment, comorbidities (urticaria, chronic rhinosinusitis with or without nasal polyps, vasculitis, etc.) are the drivers of choice among the different biological drugs. In the management of severe asthma, general practitioners (GPs) should play a central role in selecting patients and referring them to specialized centers while Scientific Societies should train GPs to appropriately recognize difficult asthma and promote public disease awareness campaigns. CONCLUSIONS: This survey which collects the point of view of allergists and clinical immunologists from Central Italy highlights that asthma control is still not measured with validated instruments. There is a general consensus that severe asthma should be managed only in dedicated centers and to this aim it is essential to encourage patient selection from a primary care setting and develop disease awareness campaigns for patients.

Implementation of GOLD consensus report in real life: results from the Velletri-Lariano (VELA) cohort.

BACKGROUND: COPD is one of the leading causes of morbidity and mortality. Pharmacotherapy improves quality of life and reduces exacerbations although low adherence with prescribed treatments may represent a barrier to optimal disease management. The first objective of this paper is to report the distribution of COPD patients according to GOLD categories, in a sample of patients from a cohort study in an area of the Latium region in Italy. The second objective is to evaluate the agreement between the distributions of severity obtained from the HCPs and the experts included in the study board (Board). METHODS: COPD patients were given a card to collect demographic and clinical data at baseline. Information in those cards was independently evaluated by HCPs and Board to include each patient into one of the four GOLD categories. RESULTS: In a sample of 187 stable COPD patients, 59% male, mean age 70 year, the distribution of GOLD categories according to the Board was: 6% A, 34% B, 2% C, and 58% D. A discrepancy in GOLD classification was observed between the study board and field-based HCPs, regarding more than 50% of the patients, with a clear trend to underestimate the frequency of patients in D level (21%) and to overestimate the frequency in C level (21%). CONCLUSIONS: These results describe for the first time the distribution of COPD patients in an Italian cohort according to the GOLD categories, with the highest frequencies in levels B and D. The misclassification from HCPs may impact the therapeutic approach and the clinical outcomes.

Revisiting the role of the mother centriole in centriole biogenesis.

Centrioles duplicate once in each cell division cycle through so-called templated or canonical duplication. SAK, also called PLK4 (SAK/PLK4), a kinase implicated in tumor development, is an upstream regulator of canonical biogenesis necessary for centriole formation. We found that overexpression of SAK/PLK4 could induce amplification of centrioles in Drosophila embryos and their de novo formation in unfertilized eggs. Both processes required the activity of DSAS-6 and DSAS-4, two molecules required for canonical duplication. Thus, centriole biogenesis is a template-free self-assembly process triggered and regulated by molecules that ordinarily associate with the existing centriole. The mother centriole is not a bona fide template but a platform for a set of regulatory molecules that catalyzes and regulates daughter centriole assembly.

Human decidual natural killer cells as a source and target of macrophage migration inhibitory factor.

The human uterine mucosa of early pregnancy is largely populated by CD56(bright) natural killer (NK) cells (uterine (u) NK cells). The specific functions of these cells are still unknown, but their interaction and response to foetal trophoblasts are thought to be important for the establishment of a successful pregnancy. The study reported herein shows that uNK cells respond to, and produce, macrophage migration inhibitory factor (MIF), a cytokine highly expressed in the human placenta and in the cyclic and pregnant endometrium. Recombinant human MIF reduced in a dose-dependent manner the cytolytic activity of purified uNK cells against K562 cells. RT-PCR, Western blot analysis and ELISA demonstrated the synthesis and secretion of the cytokine by uNK cells. Double immunofluorescence staining showed the presence of MIF in uterine CD56 + cells. Finally, neutralization of the endogenous cytokine by a polyclonal antibody resulted in a sharp increase in the cytolytic activity of uNK cells. These findings indicate the existence of a previously unrevealed paracrine and autocrine action of MIF on uNK cells and support its contribution to the immune privilege at the maternal-foetal interface.

SAK/PLK4 is required for centriole duplication and flagella development.

BACKGROUND: SAK/PLK4 is a distinct member of the polo-like kinase family. SAK-/- mice die during embryogenesis, whereas SAK+/- mice develop liver and lung tumors and SAK+/- MEFs show mitotic abnormalities. However, the mechanism underlying these phenotypes is still not known. RESULTS: Here, we show that downregulation of SAK in Drosophila cells, by mutation or RNAi, leads to loss of centrioles, the core structures of centrosomes. Such cells are able to undergo repeated rounds of cell division, but display broad disorganized mitotic spindle poles. We also show that SAK mutants lose their centrioles during the mitotic divisions preceding male meiosis but still produce cysts of 16 primary spermatocytes as in the wild-type. Mathematical modeling of the stereotyped cell divisions of spermatogenesis can account for such loss by defective centriole duplication. The majority of spermatids in SAK mutants lack centrioles and so are unable to make sperm axonemes. Finally, we show that depletion of SAK in human cells also prevents centriole duplication and gives rise to mitotic abnormalities. CONCLUSIONS: SAK/PLK4 is necessary for centriole duplication both in Drosophila and human cells. Drosophila cells tolerate the lack of centrioles and undertake mitosis but cannot form basal bodies and hence flagella. Human cells depleted of SAK show error-prone mitosis, likely to underlie its tumor-suppressor role.

Failure of pronuclear migration and repeated divisions of polar body nuclei associated with MTOC defects in polo eggs of Drosophila.

The meiotic spindle of Drosophila oocytes is acentriolar but develops an unusual central microtubule organising centre (MTOC) at the end of meiosis I. In polo oocytes, this common central pole for the two tandem spindles of meiosis II was poorly organised and in contrast to wild-type failed to maintain its associated Pav-KLP motor protein. Furthermore, the polar body nuclei failed to arrest at metaphase, and the four products of female meiosis all underwent repeated haploid division cycles on anastral spindles. This was linked to a failure to form the astral array of microtubules with which the polar body chromosomes are normally associated. The MTOC associated with the male pronucleus was also defective in polo eggs, and the sperm aster did not grow. Migration of the female pronucleus did not take place and so a gonomeric spindle could not form. We discuss these findings in relation to the known roles of polo like kinases in regulating the behaviour of MTOCs.

Disruption and phenotypic analysis of six novel genes from chromosome IV of Saccharomyces cerevisiae reveal YDL060w as an essential gene for vegetative growth.

The disruption of six novel genes (YDL059c, YDL060w, YDL063c, YDL065c, YDL070w and YDL110c), localized on the left arm of chromosome IV in Saccharomyces cerevisiae, is reported. A PCR-based strategy was used to construct disruption cassettes in which the kanMX4 dominant marker was introduced between two long flanking homology regions, homologous to the promoter and terminator sequences of the target gene (Wach et al., 1994). The disruption cassettes were used to generate homologous recombinants in two diploid strains with different genetic backgrounds (FY1679 and CEN. PK2), selecting for geneticin (G418) resistance conferred by the presence of the dominant marker kanMX4. The correctness of the cassette integration was tested by PCR. After sporulation and tetrad analysis of the heterozygous deletant diploids, geneticin-resistant haploids carrying the disrupted allele were isolated. YDL060w was shown to be an essential gene for vegetative growth. A more detailed phenotypic analysis of the non-lethal haploid deletant strains was performed, looking at cell and colony morphology, growth capability on different media at different temperatures, and ability to conjugate. Homozygous deletant diploids were also constructed and tested for sporulation. Only minor differences between parental and mutant strains were found for some deletant haploids.

Evidence of actin in the cytoskeleton of microsporidia.

Using transmission electron microscopy, immuno-electron microscopy, and biochemical techniques such as 2-D electrophoresis and immunoblotting, actin was found in all biological stages of the microsporidia Encephalitozoon hellem and Encephalitozoon cuniculi.

Centrosome inheritance in insects: fertilization and parthenogenesis.

Centrosome biogenesis is unclear, although much structural and biochemical research has been performed in several experimental systems. An alternative model to study the assembly of functional centrosomes could be the process of zygotic centrosome formation at the beginning of embryonic development. Although it seems obvious that the sperm cell provides the centrosome at fertilization, some pieces of evidence are not in line with this point of view and give controversial results. Such an analysis could provide useful information if applied to a large variety of organisms. Since insects are a highly diverse group of organisms they provide a variety of models in which to study the process of centrosome reconstitution during fertilization. Moreover, many insect species reproduce by parthenogenesis, a special mode of reproduction in which embryonic development occurs without male contribution. Studies of unfertilized parthenogenetic eggs may therefore teach us much about the process of centrosome assembly in the absence of preexisting centrioles.

Drosophila polo kinase is required for cytokinesis.

A number of lines of evidence point to a predominance of cytokinesis defects in spermatogenesis in hypomorphic alleles of the Drosophila polo gene. In the pre-meiotic mitoses, cytokinesis defects result in cysts of primary spermatocytes with reduced numbers of cells that can contain multiple centrosomes. These are connected by a correspondingly reduced number of ring canals, structures formed by the stabilization of the cleavage furrow. The earliest defects during the meiotic divisions are a failure to form the correct mid-zone and mid-body structures at telophase. This is accompanied by a failure to correctly localize the Pavarotti kinesin- like protein that functions in cytokinesis, and of the septin Peanut and of actin to be incorporated into a contractile ring. In spite of these defects, cyclin B is degraded and the cells exit M phase. The resulting spermatids are frequently binuclear or tetranuclear, in which case they develop either two or four axonemes, respectively. A significant proportion of spermatids in which cytokinesis has failed may also show the segregation defects previously ascribed to polo1 mutants. We discuss these findings in respect to conserved functions for the Polo-like kinases in regulating progression through M phase, including the earliest events of cytokinesis.

Nup154, a new Drosophila gene essential for male and female gametogenesis is related to the nup155 vertebrate nucleoporin gene.

The Nup154 gene of Drosophila encodes a protein showing similarity with known nucleoporins: rat Nup155 and yeast Nup170 and Nup157. Hypomorphic mutant alleles of Nup154 affected female and male fertility, allowing investigation of the gene function in various steps of oogenesis and spermatogenesis. Nup154 was required in testes for cyst formation, control of spermatocyte proliferation and meiotic progression. In ovaries, Nup154 was essential for egg chamber development and oocyte growth. In both the male and female germ line, as well as in several other cell types, the Nup154 protein was detected at the nuclear membrane, but was also present inside the nucleus. Intranuclear localization has not previously been described for rat Nup155 or yeast Nup170 and Nup157. In mutant egg chambers the Nup154 protein accumulated in the cytoplasm, while it was only barely detected at the nuclear envelopes. FG repeats containing nucleoporins detected with mAb414 antibody were also mislocalized to a certain extent in Nup154 mutant alleles. This suggests that Nup154 could be required for localizing other nucleoporins within the nuclear pore complex, as previously demonstrated for the yeast Nup170. On the other hand, no evident defects in lamin localization were observed, indicating that Nup155 mutations did not affect the overall integrity of the nuclear envelope. However, ultrastructural analyses revealed that in mutant cells the morphology of the nuclear envelope was altered near the nuclear pore complexes. Finally, the multiplicity of phenotypes observed in Nup154 mutant alleles suggests that this gene plays a crucial role in cell physiology.

Mechanisms of microsporidial cell division: ultrastructural study on Encephalitozoon hellem.

The mitotic process in microsporidian Encephalitozoon hellem, a known human pathogen, has been studied with the aim of elucidating some ultrastructural aspects of its nuclear division. The presence of a nuclear spindle, of "electrondense spindle plaques" associated with the nuclear envelope and of cytoplasmic double walled vesicles are reported. We suggest that these "electrondense spindle plaques" serve as foci for intranuclear and cytoplasmic microtubule arrangements, similar to the microtubule organizing centers within the centrosomes of animal cells. The extent to which the microsporidial division process is comparable with that of more familiar eukaryotes such as yeast cells is discussed.

Microtubule organization during the early development of the parthenogenetic egg of the hymenopteran Muscidifurax uniraptor.

The origin of the zygotic centrosome is an important step in developmental biology. It is generally thought that sperm at fertilization plays a central role in forming the functional centrosome which subsequently organizes the first mitotic spindle. However, this view is not applicable in the case of parthenogenetic eggs which develop without the sperm contribution. To clarify the problem of the origin of the zygotic centrosome during parthenogenetic development, we studied a hymenopteran, Muscidifurax uniraptor. Antitubulin antibody revealed that after activation several asters assembled in the egg cytoplasm. The number of asters varied in relation to the cell cycle. They became visible from anaphase of the first meiotic division and increased in number as meiosis progressed, reaching a maximum at the first mitosis. From anaphase-telophase of the first mitosis they decreased in number and were no longer found during the third mitotic division. To elucidate the nature of these asters we performed an ultrastructural study with transmission electron microscopy and immunofluorescence with antibodies against anti-gamma-tubulin and CP190. In this way we showed the presence in these asters of centrosomal components and centrioles. Our observations suggest that the cytoplasm of Muscidifurax eggs contains a pool of inactive centrosomal precursor proteins becoming able to nucleate microtubules into well-defined asters containing centrioles after activation.

gamma-Tubulin is transiently associated with the Drosophila oocyte meiotic apparatus.

Evidence of a distinct microtubule organizing center in the meiotic apparatus of the fertilized Drosophila egg is provided by means of specific antibodies. This center contained gamma-tubulin and CP190 antigens and nucleated a transient array of radial microtubules. When the eggs were incubated with the microtubule-depolymerizing drug colchicine, gamma-tubulin became undetectable in correspondence with the meiotic chromosomes, whereas it was visible near the sperm nucleus. Since the main difference between male and female microtubule organizing centers was the presence/absence of the centrioles, we propose that these organelles were mainly involved in the spatial organization of the microtubule nucleating material.

Centriole and centrosome dynamics during the embryonic cell cycles that follow the formation of the cellular blastoderm in Drosophila.

We have used immunofluorescence and electron microscopy to examine centrosome dynamics during the first postblastodermic mitoses in the Drosophila embryo. The centrosomal material, as recognized by antibodies against CP190 and gamma-tubulin, does not show the typical shape changes observed in syncytial embryos, but remains compact throughout mitosis. Centrioles, however, behave as during the syncytial mitoses, with each daughter cell inheriting two separated centrioles at the end of telophase. During interphase in epithelial cells that have a distinct G1 phase, two isolated centrioles are found, suggesting that the separation of sister centrioles is tightly coupled to a mitotic oscillator in both the "abbreviated" and the "complete" embryonic division cycles. The centrioles of the Drosophila embryo sharply differed from the sperm basal body, having a cartwheel structure with nine microtubular doublets and a central tubule. This "immature" centriolar morphology was shown to persist throughout embryonic development, clearly demonstrating that these centrioles are able to replicate despite their apparently neotenic structure.

Assembly of the zygotic centrosome in the fertilized Drosophila egg.

Zygotic centrosome assembly in fertilized Drosophila eggs was analyzed with the aid of an antiserum Rb188, previously shown to be specific for CP190, a 190 kDa centrosome-associated protein (Whitfield et al. (1988) J. Cell Sci. 89, 467-480; Whitfield et al. (1995) J. Cell Sci. 108, 3377-3387). The CP190 protein was detected in two discrete spots, associated with the anterior and posterior ends of the elongating nucleus of Drosophila spermatids. As the spermatids matured, this labelling gradually disappeared and was no longer visible in sperm dissected from spermathecae and ventral receptacles. gamma-Tubulin was also found in association with the posterior end of the sperm nucleus during spermiogenesis, but was not detected in mature sperm. This suggests that CP190 and gamma-tubulin are not present in detectable quantities in fertilizing sperm. CP190 was not detected in association with the sperm nucleus of newly fertilized eggs removed from the uterus, whereas many CP190-positive particles were associated with microtubules of the sperm aster from anaphase I to anaphase II. These particles disappeared during early telophase II and only one pair of CP190-positive spots remained visible at the microtubule focus of the sperm aster. These spots were associated with one aster through telophase, and then moved away to form two smaller asters from which the first mitotic spindle was organized. Colchicine treatment suggested that at least some CP190 protein is an integral part of the centrosome rather than merely being transported along microtubules. Centrosomal localization of the CP190 antigen was prevented by incubation of the permeabilized zygote in 20 mM EDTA.

Wolbachia-induced delay of paternal chromatin condensation does not prevent maternal chromosomes from entering anaphase in incompatible crosses of Drosophila simulans.

The behavior of parental chromosomes during the first mitosis of Drosophila simulans zygotes obtained from unidirectional incompatible crosses is described and it is demonstrated that the condensation of parental chromatin complements was asynchronous. The timing of paternal chromatin condensation appeared to be delayed in these embryos, so that condensed maternal chromosomes and entangled prophase-like paternal fibers congressed in the equatorial plane of the first metaphase spindle. At anaphase the maternal chromosomes migrated to opposite poles of the spindle, whereas the paternal chromatin lagged in the midzone of the spindle. This resulted in dramatic errors in paternal chromatin inheritance leading to the formation of embryos with aneuploid or haploid nuclei. These observations suggest that the anaphase onset of maternal chromosomes is unaffected by the improper alignment of the paternal complement. Since the first metaphase spindle of the Drosophila zygote consists of twin bundles of microtubules each holding one parental complement, we suspect that each half spindle regulates the timing of anaphase onset of its own chromosome set. In normal developing embryos, the fidelity of chromosome transmission is presumably ensured by the relative timing required to prepare parental complements for the orderly segregation that occurs during the metaphase-anaphase transition.

Patterns of microtubule assembly in taxol-treated early Drosophila embryo.

Incubation of early Drosophila embryos with low concentrations of taxol (2.3 microM) revealed a pattern of microtubule assembly that was cell-cycle dependent. Microtubule bundling was observed during the pronuclear stage after resumption of meiosis, whereas at the onset of the first mitosis the microtubules organized in astral arrays. Taxol treatment showed differential microtubule assembly properties of the egg cytoplasm. The preferential assembly site for taxol-induced asters was the ventral cortex; in the dorsal cortex only microtubule bundling occurred. This dorsal-ventral heterogeneity of the ege cortex persisted until the third or fourth nuclear cycle. Microtubules did not organize in astral arrays in the inner cytoplasm, but only in mitotic spindles. CP190 and gamma-tubulin, usually found in the centrosome of the early Drosophila embryo, were absent in taxol-induced asters. These observations suggest that the mechanism driving the assembly of taxol-induced asters is not centrosome dependent in the early Drosophila embryo.

Fertilization in Drosophila melanogaster: centrosome inheritance and organization of the first mitotic spindle.

Microtubule, chromatin, centrosome, and nuclear envelope configurations during the first division of the Drosophila melanogaster zygote were analyzed in order to investigate the organization of the first cleavage spindle and the origin of the functional centrosome. After pronuclear apposition the parental complements congress at the equatorial plane of the metaphase spindle. The chromatids, however, seem to move to the poles in two separate groups in each half spindle, mingling together during telophase, before the formation of the daughter nuclei. The spatial separation of parental complements during the first mitosis is also supported by the behavior of the nuclear envelope of female and male pronuclei. A low frequency of polyspermy is also observed during fertilization in D. melanogaster.

Meiotic spindle organization in fertilized Drosophila oocyte: presence of centrosomal components in the meiotic apparatus.

We examined spindle reorganization during the completion of meiosis in fertilized and unfertilized oocytes of Drosophila using indirect immunofluorescence and laser scanning confocal microscopy. The results defined a complex pathway of spindle assembly during resumption of meiosis, and revealed a transient array of microtubules radiating from the equatorial region of the spindle towards discrete foci in the egg cortex. A monastral array of microtubules was observed between twin metaphase II spindles in fertilized and unfertilized eggs. The microtubules originated from disk-shaped material stained with Rb188 antibody specific for an antigen associated with the centrosome of Drosophila embryos. The Drosophila egg, therefore, contains a maternal pool of centrosomal components undetectable in mature inactivated oocytes. These components nucleate microtubules in a monastral array after activation, but are unable to organize bipolar spindles.