Oscillatory Positive Expiratory Pressure in Chronic Obstructive Pulmonary Disease

Evidence-based guidance for the use of airway clearance techniques (ACT) in chronic obstructive pulmonary disease (COPD) is lacking in-part because well-established measurements of pulmonary function such as the forced expiratory volume in 1s (FEV₁) are relatively insensitive to ACT. The objective of this crossover study was to evaluate daily use of an oscillatory positive expiratory pressure (oPEP) device for 21–28 days in COPD patients who were self-identified as sputum-producers or non-sputum-producers.

COPD volunteers provided written informed consent to daily oPEP use in a randomized crossover fashion. Participants completed baseline, crossover and study-end pulmonary function tests, St. George's Respiratory Questionnaire (SGRQ), Patient Evaluation Questionnaire (PEQ), Six-Minute Walk Test and ³He magnetic resonance imaging (MRI) for the measurement of ventilation abnormalities using the ventilation defect percent (VDP).

Fourteen COPD patients, self-identified as sputum-producers and 13 COPD-non-sputum-producers completed the study. Post-oPEP, the PEQ-ease-bringing-up-sputum was improved for sputum-producers (p = 0.005) and non-sputum-producers (p = 0.04), the magnitude of which was greater for sputum-producers (p = 0.03). There were significant post-oPEP improvements for sputum-producers only for FVC (p = 0.01), 6MWD (p = 0.04), SGRQ total score (p = 0.01) as well as PEQ-patient-global-assessment (p = 0.02). Clinically relevant post-oPEP improvements for PEQ-ease-bringing-up-sputum/PEQ-patient-global-assessment/SGRQ/VDP were observed in 8/7/9/6 of 14 sputum-producers and 2/0/3/3 of 13 non-sputum-producers. The post-oPEP change in ³He MRI VDP was related to the change in PEQ-ease-bringing-up-sputum (r = 0.65, p = 0.0004) and FEV₁ (r = –0.50, p = 0.009).

In COPD patients with chronic sputum production, PEQ and SGRQ scores, FVC and 6MWD improved post-oPEP. FEV₁ and PEQ-ease-bringing-up-sputum improvements were related to improved ventilation providing mechanistic evidence to support oPEP use in COPD. Clinical Trials # NCT02282189 and NCT02282202.     

Loss of cutaneous microbial diversity during first 3 weeks of life in very low birthweight infants

Neonatal sepsis (NS) is a frequent problem in neonatal intensive care, especially in preterm and very low birthweight (VLBW) infants. The objective of the study was to characterize the cutaneous bacterial microbiome in VLBW infants treated in the neonatal intensive care unit (NICU). Non‐invasive skin microbiome specimens were taken repeatedly from 12 VLBW infants during treatment in NICU starting on the first day of life. All infants received benzylpenicillin and netilmicin during the first 1‐5 postnatal days. Samples were also collected from incubators. High cutaneous microbial diversity was present at birth in 11 of 12 of the infants, but the diversity decreased substantially after the first weeks of life in all infants regardless of their infection status. After the loss of diversity, one Staphylococcus operational taxonomic unit dominated the skin microbiome. Recovery of microbial diversity was seen in six of 12 neonates. The microbiome of incubators showed typical environmental bacterial genera. Maternal antibiotic treatment, the aetiology of the preterm birth or being born by C‐section did not appear to affect the diversity of skin microbiota at birth, and no correlation was found between cutaneous microbiome and NS.     

Characterization of intestinal invasion by Salmonella typhimurium and Salmonella dublin and effect of a mutation in the invH gene.

The relative levels of invasiveness of two bovine isolates each of Salmonella typhimurium and Salmonella dublin and of invH mutants of S. typhimurium were determined in MDCK and Int 407 cultured-cell assays and in bovine ileal loops. S. dublin was found to be significantly less invasive in cultured cells than S. typhimurium, but this difference was not observed in bovine intestines. The invH mutants exhibited a significant reduction in invasion in both cultured cells and bovine intestines. The invasive phenotypes of the strains were confirmed by fluorescent microscopy and scanning and transmission electron microscopy. The wild-type strains were observed in the laminae propriae of the intestinal villi, while in contrast the invH mutants were generally associated with the enterocyte layer. The degree of damage in the bovine ileum was related to the magnitude of the invasion. There was no difference in the amount of S. typhimurium or S. dublin recovered from the bovine ileum either with or without Peyer's patches 3 h after inoculation of the loop.     

Panfungal PCR and multiplex liquid hybridization for detection of fungi in tissue specimens

A procedure based on panfungal PCR and multiplex liquid hybridization was developed for the detection of fungi in tissue specimens. The PCR amplified the fungal internal transcribed spacer (ITS) region (ITS1-5.8S rRNA-ITS2). After capture with specific probes, eight common fungal pathogens (Aspergillus flavus, Aspergillus fumigatus, Candida albicans, Candida krusei, Candida glabrata, Candida parapsilosis, Candida tropicalis, and Cryptococcus neoformans) were identified according to the size of the amplification product on an automated sequencer. The nonhybridized products were identified by sequencing. The performance of the procedure was examined with 12 deep-tissue specimens and 8 polypous tissue biopsies from the paranasal sinuses. A detection level of 0.1 to 1 pg of purified DNA (2 to 20 CFU) was achieved. Of the 20 specimens, PCR was positive for 19 (95%), of which 10 (53%) were hybridization positive. In comparison, 12 (60%) of the specimens were positive by direct microscopy, but only 7 (35%) of the specimens showed fungal growth. Sequencing of the nonhybridized amplification products identified an infecting agent in six specimens, and three specimens yielded only sequences of unknown fungal origin. The procedure provides a rapid (within 2 days) detection of common fungal pathogens in tissue specimens, and it is highly versatile for the identification of other fungal pathogens.     

Prion protein activates and fixes complement directly via the classical pathway: implications for the mechanism of scrapie agent propagation in lymphoid tissue

C1q-deficient and complement depleted mice are highly resistant to intraperitoneal scrapie infection. The molecular mechanisms of complement involvement in scrapie pathogenesis remain unclear. Previous detailed studies have indicated mouse prion protein interactions with human C1q but the question of subsequent complement activation has remained unaddressed. In this investigation, murine prion protein, both recombinant and also from diseased tissue sources, directly activated and fixed complement via the classical but not the alternative pathway. The importance of complexed cupric ions was observed. In addition, evidence of IgG-independent C4 fixation by prion proteins was also shown. Surface plasmon resonance binding studies using variously clustered immobilized recombinant mouse prion protein indicated strong interactions with both purified mouse C1q and also mouse Factor H. Binding, especially by C1q, was dependent upon the volume of immobilized prion protein, suggesting a threshold of clustering density required to support strong interactions. Furthermore, clustered immobilized prion protein appeared capable of promoting polymerization of soluble-phase monomeric prion protein. Direct covalent attachment of complement components to prion proteins via classical pathway activation illustrates a potential mechanism underpinning their trafficking to, and subsequent propagation within, lymphoid tissues.     

Analysis of the floral transcriptome uncovers new regulators of organ determination and gene families related to flower organ differentiation in Gerbera hybrida (Asteraceae)

Development of composite inflorescences in the plant family Asteraceae has features that cannot be studied in the traditional model plants for flower development. In Gerbera hybrida, inflorescences are composed of morphologically different types of flowers tightly packed into a flower head (capitulum). Individual floral organs such as pappus bristles (sepals) are developmentally specialized, stamens are aborted in marginal flowers, petals and anthers are fused structures, and ovaries are located inferior to other floral organs. These specific features have made gerbera a rewarding target of comparative studies. Here we report the analysis of a gerbera EST database containing 16,994 cDNA sequences. Comparison of the sequences with all plant peptide sequences revealed 1656 unique sequences for gerbera not identified elsewhere within the plant kingdom. Based on the EST database, we constructed a cDNA microarray containing 9000 probes and have utilized it in identification of flower-specific genes and abundantly expressed marker genes for flower scape, pappus, stamen, and petal development. Our analysis revealed several regulatory genes with putative functions in flower-organ development. We were also able to associate a number of abundantly and specifically expressed genes with flower-organ differentiation. Gerbera is an outcrossing species, for which genetic approaches to gene discovery are not readily amenable. However, reverse genetics with the help of gene transfer has been very informative. We demonstrate here the usability of the gerbera microarray as a reliable new tool for identifying novel genes related to specific biological questions and for large-scale gene expression analysis.     

Clonal Analysis of Regulatory T Cell Defect in Patients with Autoimmune Polyendocrine Syndrome Type 1 Suggests Intrathymic Impairment

Mutations in the autoimmune regulator gene disrupt thymic T cell development and negative selection, leading to the recessively inherited polyendocrine autoimmune disease autoimmune polyendocrine syndrome type 1 (APS‐1). The patients also have a functional defect in the FOXP3⁺ regulatory T cell population, but its origin is unclear. Here, we have used T cell receptor sequencing to analyse the clonal relationship of major CD4⁺ T cell subsets in three patients and three healthy controls. The naive regulatory T cells showed little overlap with helper T cell subsets, supporting divergence in the thymus. The activated/memory regulatory T cell subset displayed more sharing with helper T cells, but was mainly recruited from the naive regulatory T cell population. These clonal patterns were very similar in both patients and controls. However, naive regulatory T cells isolated from the patients had a significantly longer T cell receptor complementarity‐determining region 3 than any other population, suggesting failure of thymic selection. These data indicate that the peripheral differentiation of regulatory T cells in APS‐1 patients is not different from that in healthy controls. Rather, the patients' naive regulatory T cells may have an intrinsic defect imprinted already in the thymus.