Microbiological changes observed over 48 weeks of treatment with inhaled liposomal ciprofloxacin in individuals with non-cystic fibrosis bronchiectasis and chronic Pseudomonas aeruginosa lung infection

ObjectivesNon-cystic fibrosis bronchiectasis (NCFBE) with Pseudomonas aeruginosa has been associated with increased pulmonary exacerbation (PEx) and mortality risk. European Respiratory Society guidelines conditionally recommend inhaled antimicrobials for persons with NCFBE, P aeruginosa and three or more PEx/year. We report microbiological results of two randomized, 48-week placebo-controlled trials of ARD-3150 (inhaled liposomal ciprofloxacin) in individuals with NCFBE with P aeruginosa and PEx history [Lancet Respir Med 2019;7:213–26].MethodsRespiratory secretions from 582 participants receiving up to six 28-day on/off treatment cycles were analysed for sputum P. aeruginosa, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus aureus and Escherichia coli densities, P. aeruginosa susceptibilities to ciprofloxacin and nine other antimicrobials, and prevalence of other bacterial opportunists. Associations between PEx risk and sputum density, antimicrobial susceptibility and opportunist prevalence changes were studied.ResultsSputum P. aeruginosa density reductions from baseline after ARD-3150 treatments ranged from 1.77 (95% CI 2.13–1.40) versus 0.54 (95% CI 0.89–0.19) log₁₀ CFU/g for placebo (second period) to 2.07 (95% CI 2.45–1.69) versus 0.70 (95% CI 1.11–0.29) log₁₀ CFU/g for placebo (fourth period) with only modest correlation between density reduction magnitude and PEx benefit. ARD-3150 (but not placebo) treatment was associated with increased P. aeruginosa ciprofloxacin MIC but not emergence of other bacterial opportunists across the study; ciprofloxacin MIC₅₀ increased from 0.5 to 1 mg/L, MIC₉₀ increased from 4 to 16 mg/L. Other antimicrobial MIC were mostly unaffected.ConclusionMicrobiological changes over 48 weeks of ARD-3150 treatment appear modest. Ciprofloxacin susceptibility (but not other antimicrobial susceptibility) decreases were observed that did not appear to preclude PEx risk reduction benefit.     

Presence of pldA and exoU in mucoid Pseudomonas aeruginosa is associated with high risk of exacerbations in non–cystic fibrosis bronchiectasis patients

ObjectivesTo explore the association between the virulence genes exoU and pldA in isolated mucoid Pseudomonas aeruginosa and the clinical outcomes of patients with non–cystic fibrosis (CF) bronchiectasis.MethodsA prospective observational cohort study was performed in the Shanghai Pulmonary Hospital from October 2012 to January 2015. We consecutively enrolled all non-CF bronchiectasis patients with mucoid P. aeruginosa isolates obtained from bronchoalveolar lavage fluid or sputum. The exposure variable was the presence of virulence gene, exoU or pldA, in the strains. The primary outcome was exacerbation of bronchiectasis. Logistic regression analysis was performed to evaluate the association between virulence genes and exacerbation.ResultsThe final analysis included 147 patients (mean (SD) age, 57.86 (11.43) years, 101 female subjects) with median (interquartile range) follow-up of 18 (13–26) months. The following factors were relative to exacerbations: body mass index ≤18.5 kg/m² (odds ratio (OR) = 5.05; 95% confidence interval (CI), 1.37–18.57), length of stay ≥8 days (OR = 2.65; 95% CI, 1.14–6.19) and positive for either virulence gene (OR = 6.80; 95% CI, 1.47–31.37). The gene-positive group had more exacerbations per year (mean 2.37, SD 2.10, n = 33 vs. mean 0.79, SD 0.83, n = 114) and a higher proportion of patients with exacerbation (31/33, 93.94% vs. 74/114, 64.91%). The proportion of patients being exoU or pldA positive increased as the exacerbation frequency of bronchiectasis increased.ConclusionsThe virulence genes exoU and pldA in mucoid P. aeruginosa are significant risk factors for exacerbations in patients with non-CF bronchiectasis.     

Induction of immune response to the 17 kDa OMPA Burkholderia cenocepacia polypeptide and protection against pulmonary infection in mice after nasal vaccination with an OMP nanoemulsion-based vaccine

Burkholderia cepacia complex (Bcc) are opportunistic bacteria associated with life-threatening illness in persons with cystic fibrosis. Once Bcc colonization is established, these antimicrobial-resistant and biofilm-forming bacteria are difficult to eradicate and are associated with increased rates of morbidity and mortality. At present, no vaccines are available to prevent the Bcc infection. There is currently a paucity of published information regarding the development of vaccines designed to prevent Burkholderia colonization. This work expands on the recent studies published by Bertot et al. [Infect Immun 75(6):2740–2752, 2007], where successful protective immune responses were generated in mice using a B. multivorans OMP-based vaccine. Here, we evaluate an experimental mucosal vaccine against Bcc using a novel mucosal adjuvant (nanoemulsion) and a novel B. cenocepacia-based OMP antigen. The OMP antigen derived from B. cenocepacia was mixed with either nanoemulsion or with PBS and delivered intranasally to CD-1 mice. Serum analysis showed robust IgG and mucosal secretory IgA immune responses in vaccinated versus control mice. The antibodies had cross-neutralizing activity against both B. cenocepacia and B. multivorans species. We found that immunized mice were protected against pulmonary colonization with B. cenocepacia. We have also identified that a 17 kDa OmpA-like protein highly conserved between Burkholderia and Ralstonia species as a new immunodominant epitope in mucosal immunization.     

Occurrence of aminoglycoside-modifying enzymes genes (aac(6′)-I and ant(2″)-I) in clinical isolates of Pseudomonas aeruginosa from Southwest Nigeria

Background

Enzymatic modification of aminoglycosides is the primary mechanism of resistance by Pseudomonas aeruginosa.

Obejectives

We investigated the occurrence and mechanism of aminoglycosides resistance in P. aeruginosa isolates from hospitals in SouthWest Nigeria.

Methods

A total of 54 consecutive, non-duplicate clinical isolates of P. aeruginosa were studied for the presence of aminoglycosides -modifying enzymes (AMEs) by PCR amplification and sequencing of genes encoding AMEs.

Results and conclusion

Two types of AME genes [aac (6′) - I and ant (2″) - I] were found in 12 isolates out of 54. Seven strains harboured one or more types of enzymes of which aac (6′) - I was the most frequently found gene (10/54 isolates, 18.5%). None of the isolates investigated in this study were positive for aph, aac (3) and aac (6″) - II genes. Prevalence of P. aeruginosa producing AME genes in this study may suggest aminoglycosides use in Nigeria. This study highlights need for functional antimicrobial surveillance system in Nigeria.     

Clinical experience with α-galactosylceramide (KRN7000) in patients with advanced cancer and chronic hepatitis B/C infection

For over a century, research has sought ways to boost the immune system in order to eradicate tumors and viruses that exist after escaping immunosurveillance. For the treatment of cancer and hepatitis immunotherapeutic strategies have overall had limited clinical success. An urgent need exists therefore to introduce more effective therapeutic approaches. Invariant (i)NKT cells constitute a conserved T lymphocyte lineage with dominant immunoregulatory, antitumor and antiviral effector cell properties. iNKT specifically recognize the glycolipid α-galactosylceramide in the context of CD1d resulting in their activation. Activated iNKT can promote the development of a long-lasting Th1 biased proinflammatory immune response as demonstrated in multiple tumor-metastasis and viral infection models. Here, we will provide a brief overview of the preclinical data of α-galactosylceramide that formed the basis for subsequent clinical trials in patients with advanced cancer and chronic hepatitis B/C, and elaborate on our own clinical experience with α-galactosylceramide in these patient groups.     

Effect of chlorine on cultivability of Shiga toxin producing Escherichia coli (STEC) and β-lactamase genes carrying E. coli and Pseudomonas aeruginosa

The worldwide spread of toxin-producing and multi-drug resistant bacteria in water, food and the environment is considered a major threat to human health. Drinking water quality is controlled by inspection of fecal indicators presence whereby viable contaminants will be efficiently reduced by chlorination which is a common process for disinfection. However, the all-out efficiency is arguable, because bacterial regrowth has been documented after disinfection. In this study, we investigated the stability of Shiga toxin producing Escherichia coli (STEC) and β-lactamase expressing E. coli and Pseudomonas aeruginosa isolates, both equipped with multiple or single β-lactamase resistance genes. The aim of the study was to analyze the efficiency of chlorine (Cl₂) disinfection against shigatoxigenic or β-lactamase producing bacteria. Cl₂ reacts with the bacterial cells after first contact. Counts of antibiotic resistant E. coli were lower after short than upon extended Cl₂ treatment. P. aeruginosa counts decreased moderately upon 15–60 min treatment with 1.2 mg Cl₂/l, while cells adapted to tap water were not cultivable anymore. We assume that the bacterial physiology changed to a temporary non-cultivatable state at first Cl₂ contact followed by resuscitation of some cells at later stages. STEC viability went down continuously at low Cl₂ concentrations and these toxigenic E. coli isolates exhibited slightly increased stability to Cl₂ treatment compared with non-toxigenic E. coli. Controlling the efficiency of disinfection, realistic counts of cultivatable cells are achieved after extended Cl₂ action.     

A novel VIM‐type metallo‐beta‐lactamase (VIM‐14) in a Pseudomonas aeruginosa clinical isolate from a neonatal intensive care unit

A Pseudomonas aeruginosa highly resistant to carbapenems was isolated in a neonatal intensive care unit in Palermo, Italy. The strain was found to carry a novel VIM‐type enzyme, classified as VIM‐14. The novel enzyme differs from VIM‐4 in a G31S mutation. VIM‐14 was harboured in a class 1 integron with a new organization. The integron carried the genes aac7, blaVIM‐14, blaOXA‐20 and aac4 in that order.     

Distinct Contributions of Interleukin-1α (IL-1α) and IL-1β to Innate Immune Recognition of Pseudomonas aeruginosa in the Lung

The bacterial pathogen Pseudomonas aeruginosa causes acute infections associated with significant morbidity and mortality. P. aeruginosa elicits strong innate immune responses in immunocompetent hosts, and the resulting recruitment of neutrophils to the site of infection is necessary for bacterial clearance. P. aeruginosa lipopolysaccharide and flagellin are recognized by extracellular Toll-like receptors, but the most rapid responses to infection occur when cytosolic receptors sense flagellin or type 3 secretion system (T3SS) structural proteins. The subsequent activation of the NLRC4 inflammasome and caspase-1 generates an interleukin-1β (IL-1β) signal that is required for the rapid neutrophilic response. A T3SS effector, exotoxin U (ExoU), can inhibit activation of the NLRC4 inflammasome and caspase-1. Thus, our observation that IL-1 receptor (IL-1R)-mediated signals were still required to initiate a response to ExoU-producing bacteria was unexpected. As both IL-1α and IL-1β signal via the IL-1R, we examined immune responses in mice lacking either of these cytokines. IL-1β-deficient mice responded to ExoU-producing P. aeruginosa bacteria similarly to wild-type animals; however, IL-1α-deficient mice had an attenuated immune response. The situation was reversed following infections by ExoU-negative bacteria: here, IL-1α was dispensable for neutrophil recruitment, while IL-1β was required. IL-1α secretion by macrophages infected with ExoU-producing P. aeruginosa isolates was independent of both caspase-1 and caspase-11. This study documents distinct roles for IL-1α and IL-1β in the response to P. aeruginosa infection as a function of the T3SS effectors produced by the infecting strain. The redundancy of these two cytokines nonetheless allows the infected host to mount a response to ExoU-positive and -negative bacterial isolates.     

Surface IgM λ light chain is involved in the binding and infection of infectious bursal disease virus (IBDV) to DT40 cells

Infectious bursal disease virus (IBDV) is an important immunosuppressive virus in chickens. Surface immunoglobulin M (sIgM)-bearing B lymphocytes act as the major targets of IBDV in the bursa of Fabricius, and sIgM may function as one of the membrane binding sites responsible for IBDV infection. Recently, using the virus overlay protein binding assay, the chicken λ light chain of sIgM was identified to specifically interact with IBDV in a virulence-independent manner in vitro. To further investigate sIgM λ light chain-mediated IBDV binding and infection in pre-B cells, the cell line DT40, which is susceptible to both pathogenic and attenuated IBDV, was used. Based on the RNA interference strategy, the DT40 cell line whose λ light chain of sIgM was stably knocked down, herein termed DT40LKD, was generated by the genomic integration of a specific small hairpin RNA and a green fluorescence protein co-expression construct. Flow cytometry analysis indicated that the binding of IBDV to DT40LKD cells was significantly reduced due to the loss of sIgM λ light chain. In particular, reduced viral replication was observed in IBDV-incubated DT40LKD cells, and no viral release into cell culture medium was detected by the IBDV rapid diagnostic strips. In addition, the rescue of sIgM λ light chain expression restored viral binding and replication in DT40LKD cells. These results show that sIgM λ light chain appears to be beneficial for IBDV attachment and infection, suggesting that sIgM acts as a binding site involved in IBDV infection.     

Correlation of virus replication,cytokine (TNF-α and IL-1) producing cells,neuronal necrosis and inflammation after intranasal infection of mice with herpes simplex virus strains of different virulence

Summary The number of TNF- and IL-1 producing cells was investigated during the acute replication phase of herpes simplex virus (HSV) in trigeminal ganglia after intranasal infection with strains of different virulence. The highly virulent strain WAL replicated strongly and induced many cytokine producing cells early in the ganglia. The low virulent strain HFEM replicated less, only few cytokine producing cells were detected late. The thymidine-kinase negative (TK^–) virus 1301 did not replicate but produced some lymphocytic inflammation. The higher the virulence of strains of HSV-1 or -2 was, the stronger was the extent of histopathological lesions; moreover, a dissociation in time between replication and cellular reaction (granulocytic and lymphocytic) could be observed after infection with strains HFEM and TK^– virus 1301. CD4 and CD8 positive cells could be detected mainly at the rim of necrotic areas, TNF- and IL-1 producing cells, however, were scattered throughout the ganglia.     

The stimulatory effects of caffeine with oseltamivir (Tamiflu) on light–dark behavior and open-field behavior in mice

Abnormal behaviors and death associated with the use of oseltamivir (Tamiflu^®) have emerged as a major issue in influenza patients taking the drug. Here, we investigated the mechanisms underlying the effects of oseltamivir on the behavior of mice using light–dark and open-field preference tests. Oseltamivir (75 and 150 mg/kg, intraperitoneally (i.p.)) alone affected neither time spent in the open area in the light–dark preference test nor ambulation in the open-field test at 2 h post-injection. However, a non-selective adenosine A₁/A₂ receptor antagonist, caffeine (10 mg/kg, i.p.) in combination with oseltamivir (150 mg/kg, i.p.) increased time spent in the open area in the light–dark preference test. This enhancement was not inhibited by a benzodiazepine receptor antagonist, flumazenil (10–20 mg/kg, subcutaneously (s.c.)). Enhancement of ambulation in the open-field test was also observed when caffeine (10 mg/kg, i.p.) was combined with oseltamivir (150 mg/kg, i.p.). This enhancement was inhibited by a dopamine D₂ receptor antagonist, haloperidol (0.1 mg/kg, s.c.). Furthermore, an adenosine A₂ receptor antagonist, SCH58261 (3 mg/kg, i.p.) in combination with oseltamivir (150 mg/kg, i.p.) increased ambulation in the open-field test, while an adenosine A₁ receptor antagonist, DPCPX (1–3 mg/kg, i.p.) did not. These findings suggest that the actions of oseltamivir may involve the dopamine and adenosine systems. Our findings suggest that due to the interaction between central blockade of adenosine A₂ receptors by caffeine, and oseltamivir-induced behavioral changes, patients being treated with oseltamivir should be closely monitored.     

In vivo targeting and positron emission tomography imaging of tumor vasculature with (66)Ga-labeled nano-graphene

The goal of this study was to employ nano-graphene for tumor targeting in an animal tumor model, and quantitatively evaluate the pharmacokinetics and tumor targeting efficacy through positron emission tomography (PET) imaging using (66)Ga as the radiolabel. Nano-graphene oxide (GO) sheets with covalently linked, amino group-terminated six-arm branched polyethylene glycol (PEG; 10 kDa) chains were conjugated to NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid, for (66)Ga-labeling) and TRC105 (an antibody that binds to CD105). Flow cytometry analyses, size measurements, and serum stability studies were performed to characterize the GO conjugates before in vivo investigations in 4T1 murine breast tumor-bearing mice, which were further validated by histology. TRC105-conjugated GO was specific for CD105 in cell culture. (66)Ga-NOTA-GO-TRC105 and (66)Ga-NOTA-GO exhibited excellent stability in complete mouse serum. In 4T1 tumor-bearing mice, these GO conjugates were primarily cleared through the hepatobiliary pathway. (66)Ga-NOTA-GO-TRC105 accumulated quickly in the 4T1 tumors and tumor uptake remained stable over time (3.8 ± 0.4, 4.5 ± 0.4, 5.8 ± 0.3, and 4.5 ± 0.4 %ID/g at 0.5, 3, 7, and 24 h post-injection respectively; n = 4). Blocking studies with unconjugated TRC105 confirmed CD105 specificity of (66)Ga-NOTA-GO-TRC105, which was corroborated by biodistribution and histology studies. Furthermore, histological examination revealed that targeting of NOTA-GO-TRC105 is tumor vasculature CD105 specific with little extravasation. Successful demonstration of in vivo tumor targeting with GO, along with the versatile chemistry of graphene-based nanomaterials, makes them suitable nanoplatforms for future biomedical research such as cancer theranostics.     

Low α(2)β(1) integrin function enhances the proliferation of fibroblasts from patients with idiopathic pulmonary fibrosis by activation of the β-catenin pathway

Idiopathic pulmonary fibrosis (IPF) is a progressive and incurable fibroproliferative disorder characterized by unrelenting proliferation of fibroblasts and their deposition of collagen within alveoli, resulting in permanently scarred, nonfunctional airspaces. Normally, polymerized collagen suppresses fibroblast proliferation and serves as a physiological restraint to limit fibroproliferation after tissue injury. The IPF fibroblast, however, is a pathologically altered cell that has acquired the capacity to elude the proliferation-suppressive effects of polymerized collagen. The mechanism for this phenomenon remains incompletely understood. Here, we demonstrate that expression of α(2)β(1) integrin, a major collagen receptor, is pathologically low in IPF fibroblasts interacting with polymerized collagen. Low integrin expression in IPF fibroblasts is associated with a failure to induce PP2A phosphatase activity, resulting in abnormally high levels of phosphorylated (inactive) GSK-3β and high levels of active β-catenin in the nucleus. Knockdown of β-catenin in IPF fibroblasts inhibits their ability to proliferate on collagen. Interdiction of α(2)β(1) integrin in control fibroblasts reproduces the IPF phenotype and leads to the inability of these cells to activate PP2A, resulting in high levels of phosphorylated GSK-3β and active β-catenin and in enhanced proliferation on collagen. Our findings indicate that the IPF fibroblast phenotype is characterized by low α(2)β(1) integrin expression, resulting in a failure of integrin to activate PP2A phosphatase, which permits inappropriate activation of the β-catenin pathway.     

7 Mb de novo deletion within 8q21 in a patient with distal arthrogryposis type 2B (DA2B)

We report on a 7 11/12 years old male patient with normal mental development, club feet, ulnar deviation and mild camptodactyly as well as facial dysmorphism including high forehead, small mouth, broad nasal bridge, epicanthus, high palate, brachycephalus, short neck, and dysplastic ears consistent with distal arthrogryposis type 2B (DA2B). Mutational analysis of the genes MYH3, TNNI2, TNNT3 and TPM2, known to cause DA2B revealed no apparent disease causing mutation. Molecular karyotyping using a 250?K SNP array revealed a heterozygous de novo 7?Mb deletion of 8q21.11-8q21.13 containing 23 genes. Prioritisation of possible candidate genes using the bioinformatics tool ENDEAVOUR revealed three favoured genes, HEY1, FABP5 and FABP4 as potential causes of the phenotype. We propose that the 8q21 region contains a further locus which contributes to the genetically heterogeneous DA2B.     

Growth characteristics in mammary gland tissue cultures from mice of a noncancer lineage (D) associated with the addition of androsterone and estradiol 17β to the nutritive medium

Summary The author cultured the tissues of the mammary gland obtained from 40 mice (aged from 2 to 6 months) of noncancer lineage (D). Androsterone was introduced into the fluid nutritive medium in the first series of experiments, and estradiol 17—in the second. The effect of the action of androsterone and estradiol 17 was sufficiently convincing only when culturing the tissues of the virgin mice (according to the number of the growing epithelial cultures). The effect of androsterone and estradiol on the growth intensity and the morphology of the epithelial cultures was reflected in a principally similar way both in the virgin and parous mice, the only difference being that these changes were more clear-cut in the experiment with estradiol 17. The effect of the hormones was manifest in the increased number of mitoses, including multipolar (possible also of amitoses), in the formation of the multinuclear cells, the polymorphism of the cells and nuclei, the greater width of the membrane in the series of experiments with estradiol 17 (in comparison with control and the series of experiments with androsterone). The number of the growing connective tissue cultures of the mammary gland were the same both in the control and in the experimental series, in the group of virgin and in the group of parous animals. Here the effect of estradiol 17 and androsterone was manifested in the appearance of a large number of multinuclear cells, especially in the experiment with estradiol 17.(Presented by Active Member of the Acad. Med. Sci., USSR A. I. Serebrov) Translated from Byulleten' Èksperimental'noi Biologii i Meditsiny, Vol. 49, No. 6, pp. 92–97, June, 1960