Body mass index and nutritional intake following Elexacaftor/Tezacaftor/Ivacaftor modulator therapy in adults with cystic fibrosis.

BACKGROUND: Elexacaftor/Tezacaftor/Ivacaftor (ETI) modulator therapy is often associated with increased body mass index (BMI) in people with cystic fibrosis (CF). This is thought to reflect improved clinical stability and increased appetite and nutritional intake. We explored the change in BMI and nutritional intake following ETI modulator therapy in adults with CF. METHODS: Dietary intake, measured with myfood24®, and BMI were collected from adults with CF at baseline and follow-up as part of an observational study. Changes in BMI and nutritional intake in participants who commenced ETI therapy between time points were assessed. To contextualize findings, we also assessed changes in BMI and nutritional intake between study points in a group on no modulators. RESULTS: In the pre and post ETI threapy group (n = 40), BMI significantly increased from 23.0 kg/m2 (IQR 21.4, 25.3) at baseline to 24.6 kg/m2 (IQR 23.0, 26.7) at follow-up (p<0.001), with a median of 68 weeks between time points (range 20-94 weeks) and median duration of ETI therapy was 23 weeks (range 7-72 weeks). There was a significant decrease in energy intake from 2551 kcal/day (IQR 2107, 3115) to 2153 kcal/day (IQR 1648, 2606), p<0.001. In the no modulator group (n = 10), BMI and energy intake did not significantly change between time points (p>0.05), a median of 28 weeks apart (range 20-76 weeks). CONCLUSIONS: These findings tentatively suggest that the increase in BMI with ETI therapy may not simply be attributable to an increase in oral intake. Further exploration into the underlying aetiology of weight gain with ETI therapy is needed.

Impact of a new hospital with close to 100% single-occupancy rooms on environmental contamination and incidence of vancomycin-resistant Enterococcus faecium colonization or infection: a genomic surveillance study.

BACKGROUND: Vancomycin-resistant Enterococcus faecium (VRE) is a leading cause of nosocomial infection, driven by its ability to spread between patients and persist in the hospital environment. AIM: To investigate the impact of a long-established cardiothoracic hospital moving to new premises with close to 100% single-occupancy rooms on the rates of environmental contamination and infection or colonization by VRE. METHODS: Prospective environmental surveillance for VRE was conducted at five time-points between April and November 2019, once in the original building, and four times in the new building. Incidence rate ratios (IRRs) of VRE infection/colonization were determined for the one-year period before and after the hospital move, and compared to a nearby hospital. FINDINGS: In the original location, the first environmental screen found 29% VRE positivity. The following four screens in the new location showed a significant reduction in positivity (1-6%; P<0.0001). The VRE infection/colonization rates were halved in the new location (IRR: 0.56; 95% confidence interval: 0.38-0.84), compared to the original location, contrasting with an increase in a nearby hospital (1.62; 1.17-2.27) over the same time-period. Genomic analysis of the environmental isolates was consistent with reduced transmission in the new hospital. CONCLUSION: The use of single-occupancy rooms was associated with reduced environmental contamination with VRE, and lower transmission and isolation of VRE from clinical samples. The cost-effectiveness of single-occupancy room hospitals in reducing healthcare-associated infections should be reassessed in the context of operational costs of emerging pandemic and increasing antimicrobial resistance threats.

Diabetes is associated with increased burden of gastrointestinal symptoms in adults with cystic fibrosis.

BACKGROUND: Individuals with diabetes mellitus (DM) are known to frequently experience gastrointestinal (GI) symptoms. In contrast, the impact of cystic fibrosis-related diabetes (CFRD) on accentuating GI symptoms in people with cystic fibrosis (pwCF) is unknown. We sought to examine this. METHODS: Abdominal symptoms were measured using the validated CF-specific GI symptom questionnaire - CFAbd-Score© - as part of a multicentre cohort study in pancreatic insufficient adults with CF, not on cystic fibrosis transmembrane conductance regulator (CFTR) modulators. The CFAbd-Score total score (0-100pts), its 5 domains, alongside nine specific GI symptoms associated with DM, were compared between the CFRD and non-CFRD groups. RESULTS: 27 (31%) and 61 (69%) participants with CF were recruited in the CFRD and non-CFRD groups respectively. Total CFAbd-Score and the two domains: gastroesophageal reflux disease and disorders of appetite were significantly higher in the CFRD group compared to the non-CFRD group (p<0.05), with the mean total CFAbd-Score being 25.4 ± 2.5 and 18.4 ± 1.5 in the CFRD and non-CFRD groups respectively. Among the nine GI symptoms commonly reported as elevated in DM, bloating and nausea were significantly more common in individuals with CFRD compared to those without (p<0.05). CONCLUSIONS: Individuals with CFRD overall, have a higher GI symptom burden, according to CFAbd-Scores. Specifically, they experience significantly more bloating and nausea. Close monitoring and further research is needed to better understand and manage GI symptoms in this group.

Cystic Fibrosis-Related Gut Dysbiosis: A Systematic Review.

BACKGROUND AND AIMS: Cystic Fibrosis (CF) is associated with gut dysbiosis, local and systemic inflammation, and impaired immune function. Gut microbiota dysbiosis results from changes in the complex gut milieu in response to CF transmembrane conductance regulator (CFTR) dysfunction, pancreatic malabsorption, diet, medications, and environmental influences. In several diseases, alteration of the gut microbiota influences local and systemic inflammation and disease outcomes. We conducted a systematic review of the gut microbiota in CF and explored factors influencing dysbiosis. METHODS: An electronic search of three databases was conducted in January 2019, and re-run in June 2021. Human, animal, and in vitro studies were included. The primary outcome was differences in the gut microbiota between people with CF (pwCF) and healthy controls. Secondary outcomes included the relationship between the gut microbiota and other factors, including diet, medication, inflammation, and pulmonary function in pwCF. RESULTS: Thirty-eight studies were identified. The literature confirmed the presence of CF-related gut dysbiosis, characterized by reduced diversity and several taxonomic changes. There was a relative increase of bacteria associated with a pro-inflammatory response coupled with a reduction of those considered anti-inflammatory. However, studies linking gut dysbiosis to systemic and lung inflammation were limited. Causes of gut dysbiosis were multifactorial, and findings were variable. Data on the impact of CFTR modulators on the gut microbiota were limited. CONCLUSIONS: CF-related gut dysbiosis is evident in pwCF. Whether this influences local and systemic disease and is amenable to interventions with diet and drugs, such as CFTR modulators, requires further investigation.

Microbiological evaluation of UV disinfection effectiveness in a specialist cystic fibrosis clinic.

The aim of the study was to evaluate the impact of manual cleaning and manual cleaning followed by Ultraviolet-C disinfection on the colony forming units of bacteria retrievable from equipment and surfaces within clinic rooms following a CF outpatient encounter. While UV disinfection has proven to be effective within general healthcare settings, it has not been evaluated in a CF centre. Microbiological sampling was performed following outpatient encounters involving 11 adult patients with CF and chronic infection with P.aeruginosa, MRSA or E. coli ESBL. The results of this study suggest that manual cleaning followed by UV-C disinfection is more effective than manual cleaning alone at reducing environmental contamination within a CF clinic and that UV-C isinfection is likely to reduce the risk of fomite transmission in the CF outpatient setting.

A Rapid Unraveling of the Activity and Antibiotic Susceptibility of Mycobacteria.

The development of antibiotic-resistant bacteria is a worldwide health-related emergency that calls for new tools to study the bacterial metabolism and to obtain fast diagnoses. Indeed, the conventional analysis time scale is too long and affects our ability to fight infections. Slowly growing bacteria represent a bigger challenge, since their analysis may require up to months. Among these bacteria, Mycobacterium tuberculosis, the causative agent of tuberculosis, has caused more than 10 million new cases and 1.7 million deaths in 2016 only. We employed a particularly powerful nanomechanical oscillator, the nanomotion sensor, to characterize rapidly and in real time tuberculous and nontuberculous bacterial species, Mycobacterium bovis bacillus Calmette-Guérin and Mycobacterium abscessus, respectively, exposed to different antibiotics. Here, we show how high-speed and high-sensitivity detectors, the nanomotion sensors, can provide a rapid and reliable analysis of different mycobacterial species, obtaining qualitative and quantitative information on their responses to different drugs. This is the first application of the technique to tackle the urgent medical issue of mycobacterial infections, evaluating the dynamic response of bacteria to different antimicrobial families and the role of the replication rate in the resulting nanomotion pattern. In addition to a fast analysis, which could massively benefit patients and the overall health care system, we investigated the real-time responses of the bacteria to extract unique information on the bacterial mechanisms triggered in response to antibacterial pressure, with consequences both at the clinical level and at the microbiological level.

Autologous dendritic cells prolong allograft survival through Tmem176b-dependent antigen cross-presentation.

The administration of autologous (recipient-derived) tolerogenic dendritic cells (ATDCs) is under clinical evaluation. However, the molecular mechanisms by which these cells prolong graft survival in a donor-specific manner is unknown. Here, we tested mouse ATDCs for their therapeutic potential in a skin transplantation model. ATDC injection in combination with anti-CD3 treatment induced the accumulation of CD8(+) CD11c(+) T cells and significantly prolonged allograft survival. TMEM176B is an intracellular protein expressed in ATDCs and initially identified in allograft tolerance. We show that Tmem176b(-/-) ATDCs completely failed to trigger both phenomena but recovered their effect when loaded with donor peptides before injection. These results strongly suggested that ATDCs require TMEM176B to cross-present antigens in a tolerogenic fashion. In agreement with this, Tmem176b(-/-) ATDCs specifically failed to cross-present male antigens or ovalbumin to CD8(+) T cells. Finally, we observed that a Tmem176b-dependent cation current controls phagosomal pH, a critical parameter in cross-presentation. Thus, ATDCs require TMEM176B to cross-present donor antigens to induce donor-specific CD8(+) CD11c(+) T cells with regulatory properties and prolong graft survival.

Rapamycin and mTOR-independent autophagy inducers ameliorate toxicity of polyglutamine-expanded huntingtin and related proteinopathies.

The formation of intra-neuronal mutant protein aggregates is a characteristic of several human neurodegenerative disorders, like Alzheimer's disease, Parkinson's disease (PD) and polyglutamine disorders, including Huntington's disease (HD). Autophagy is a major clearance pathway for the removal of mutant huntingtin associated with HD, and many other disease-causing, cytoplasmic, aggregate-prone proteins. Autophagy is negatively regulated by the mammalian target of rapamycin (mTOR) and can be induced in all mammalian cell types by the mTOR inhibitor rapamycin. It can also be induced by a recently described cyclical mTOR-independent pathway, which has multiple drug targets, involving links between Ca(2+)-calpain-G(salpha) and cAMP-Epac-PLC-epsilon-IP(3) signalling. Both pathways enhance the clearance of mutant huntingtin fragments and attenuate polyglutamine toxicity in cell and animal models. The protective effects of rapamycin in vivo are autophagy-dependent. In Drosophila models of various diseases, the benefits of rapamycin are lost when the expression of different autophagy genes is reduced, implicating that its effects are not mediated by autophagy-independent processes (like mild translation suppression). Also, the mTOR-independent autophagy enhancers have no effects on mutant protein clearance in autophagy-deficient cells. In this review, we describe various drugs and pathways inducing autophagy, which may be potential therapeutic approaches for HD and related conditions.

Functional coupling of FcgammaRI to nicotinamide adenine dinucleotide phosphate (reduced form) oxidative burst and immune complex trafficking requires the activation of phospholipase D1.

Immunoglobulin G (IgG) receptors (FcgammaRs) on myeloid cells are responsible for the internalization of immune complexes. Activation of the oxidase burst is an important component of the integrated cellular response mediated by Fc receptors. Previous work has demonstrated that, in interferon-gamma-primed U937 cells, the high-affinity receptor for IgG, FcgammaRI, is coupled to a novel intracellular signaling pathway that involves the sequential activation of phospholipase D (PLD), sphingosine kinase, and calcium transients. Here, it is shown that both known PLD isozymes, PLD1 and PLD2, were present in these cells. With the use of antisense oligonucleotides to specifically reduce the expression of either isozyme, PLD1, but not PLD2, was found to be coupled to FcgammaRI activation and be required to mediate receptor activation of sphingosine kinase and calcium transients. In addition, coupling of FcgammaRI to activation of the nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH) oxidase burst was inhibited by pretreating cells with 0.3% butan-1-ol, indicating an absolute requirement for PLD. Furthermore, use of antisense oligonucleotides to reduce expression of PLD1 or PLD2 demonstrated that PLD1 is required to couple FcgammaRI to the activation of NADPH oxidase and trafficking of internalized immune complexes for degradation. These studies demonstrate the critical role of PLD1 in the intracellular signaling cascades initiated by FcgammaRI and its functional role in coordinating the response to antigen-antibody complexes.

Lysosomal routing of Fc gamma RI from early endosomes requires recruitment of tyrosine kinases.

The high-affinity receptor for immunoglobulin G (Fc gamma RI) plays a central role in the clearance of immune complexes by mediating their internalization and delivery to lysosomes. In monocytic U937 cells, receptor internalization is independent of tyrosine kinase activity. However, the tyrosine kinase inhibitor, genistein, prevents further progress of the receptor to lysosomes and traps it in a sub-plasma membrane early endosome. Similarly, Fc gamma RI expressed in COS cells is able to internalize immune complexes but is unable to translocate to lysosomes. This suggests that Fc gamma RI, whose cytoplasmic tail is devoid of known signalling motifs, must recruit tyrosine kinases via its gamma-chain to achieve lysosomal delivery. We show that a chimera of the extracellular domain of Fc gamma RI and the cytoplasmic tail of the gamma-chain is both internalized and efficiently trafficked to lysosomes. Our study suggests that a key function of the gamma-chain is recruitment of tyrosine kinases to initiate the intracellular signalling pathways required to target Fc gamma RI following immune complex aggregation to lysosomes and not to initiate endocytosis per se.

FcgammaRI coupling to phospholipase D initiates sphingosine kinase-mediated calcium mobilization and vesicular trafficking.

Aggregation of receptors specific for the constant region of immunoglobulin G activates a repertoire of monocyte responses that can lead ultimately to targeted cell killing via antibody-directed cellular cytotoxicity. The high affinity receptor, FcgammaRI, contains no recognized signaling motif in its cytoplasmic tail but rather utilizes the gamma-chain of FcepsilonRI as an accessory molecule to recruit tyrosine kinases for signal transduction. We show here that, in a human monocytic cell line primed with interferon-gamma, FcgammaRI mobilizes intracellular calcium stores using a novel pathway that involves tyrosine kinase coupling to phospholipase D and resultant downstream activation of sphingosine kinase. Moreover, FcgammaRI is not coupled to phospholipase C; hence, calcium release from intracellular stores occurred in the absence of any measurable rise in inositol triphosphate. Finally, as this novel activation pathway is also shown to be responsible for mediating the vesicular trafficking of internalized immune complexes for degradation, it is likely to play a key role in controlling intracellular events triggered by FcgammaRI.

A molecular switch changes the signalling pathway used by the Fc gamma RI antibody receptor to mobilise calcium.

BACKGROUND: Leukocytes express Fc gamma receptors, which are specific for the constant region of immunoglobulin G. Aggregation of these receptors activates a repertoire of responses that can lead to targeted cell killing by antibody-directed cellular cytotoxicity. The nature of the myeloid response to Fc gamma receptor aggregation is highly variable and depends on the maturation state of the cell, but little is known about the signalling mechanisms underlying this variability. RESULTS: We show here that differentiation of a monocytic cell line, U937, to a more macrophage phenotype resulted in an absolute and fundamental switch in the nature of the phospholipid signalling pathway recruited following Fc gamma receptor aggregation. In cytokine-primed monocytes, aggregation of the high-affinity receptor Fc gamma RI resulted in the activation of phospholipase D and sphingosine kinase, which in turn led to the transient release of stored calcium; these effects were mediated by the gamma chain, an Fc gamma RI accessory protein. In contrast, in cells differentiated to a more macrophage type, aggregation of Fc gamma RI resulted in the Fc gamma RIIa-mediated activation of phospholipase C, and the resulting calcium response was prolonged as calcium entry was stimulated. CONCLUSIONS: The switch in Fc gamma RI signalling pathways upon monocyte differentiation is mediated by a switch in the accessory molecule recruited by Fc gamma RI, which lacks its own intrinsic signal transduction motif. As many immune receptors have separate polypeptide chains for ligand binding and signal transduction (allowing a similar switch in signalling pathways), the mechanism described here is likely to be widely used.

Fcgamma receptor I activation triggers a novel Ca2+-activated current selective for monovalent cations in the human monocytic cell line, U937.

Previous reports have suggested that receptors for immunoglobulin G (IgG), FcgammaRs, directly activate a nonselective cation channel (Young, J. D.-E., Unkeless, J. C., Young, T. M., Mauro, A., and Cohn, Z. A. (1983) Nature 306, 186-189; Nelson, D. J., Jacobs, E. R., Tang, J. M., Zeller, J. M., and Bone, R. C. (1985) J. Clin. Invest. 76, 500-507). To investigate the mechanisms underlying membrane conductance changes following human high affinity (FcgammaRI) receptor activation, we have used the human monocytic cell line U937 and combined conventional whole cell patch-clamp recordings with single cell fura-2 Ca2+ measurements. Using a K+-free internal solution, antibody cross-linking of IgG-occupied FcgammaRI activated an inward current at negative potentials, whose amplitude and time course mirrored the concomitant rise in intracellular Ca2+. Current-voltage relationships, obtained under different ionic conditions, revealed a monovalent cation-selective conductance that, under physiological conditions, would result in Na+ influx. Noise analysis of current recordings indicated a single channel conductance of 18 picosiemens and a mean opening time of 4.5 ms. This current was also activated by rises in intracellular Ca2+ induced by ionomycin (3 microM) or thapsigargin (1 microM). Addition of the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid to the intracellular medium abolished any channel activation by ionomycin, FcgammaRI, or the low affinity receptor, FcgammaRII. These results demonstrate that FcgammaRI activation triggers a novel Ca2+-activated channel selective for monovalent cations and that neither FcgammaRI nor FcgammaRII can directly activate a channel.

Temperature-dependent block of capacitative Ca2+ influx in the human leukemic cell line KU-812.

The mechanism by which depletion of intracellular Ca2+ stores activates Ca2+ influx is not understood. We recently showed that primaquine, an inhibitor of vesicular transport, blocks the activation of the calcium release-activated calcium current (ICRAC) in rat megakaryocytes (Somasundaram, B., Norman, J. C., and Mahaut-Smith, M. P. (1995) Biochem. J. 309, 725-729). Since it is well established that vesicular transport is temperature-sensitive, we have investigated the effect of temperature on both the activation and maintenance of store-mediated Ca2+ and Mn2+ influx in the human leukemic cell line KU-812 using a combination of whole cell ICRAC recordings and measurements of Mn2+ photoquench of fura-2. Activation of ICRAC was temperature-sensitive, showing a nonlinear reduction when the temperature was lowered from 27 to 17 degrees C with an abrupt change at 21-22 degrees C and complete inhibition at 17 degrees C. Once activated, ICRAC also displayed an abrupt reduction at 21-22 degrees C but was not completely blocked even when the temperature was reduced to 14 degrees C, suggesting that at least one of the temperature-sensitive components is exclusively involved in ICRAC activation. Activation of store-mediated Mn2+ influx also showed similar nonlinear temperature sensitivity and complete inhibition at 19 degrees C. However, in contrast to ICRAC measurements, lowering the temperature following maximal activation of the influx pathway at 37 degrees C did not result in any detectable residual Mn2+ entry below 19 degrees C. We conclude that the mechanism of store-mediated Ca2+ influx involves temperature-dependent steps in both its maintenance and activation, suggesting dependence on a lipid membrane environment.

Differentiation of the human monocytic cell line U937 results in an upregulation of the calcium release-activated current, ICRAC.

1. Single cell fura-2 fluorescence measurements and whole-cell patch clamp recordings were used to investigate the effects of macrophage-like differentiation, induced by dibutyryl cAMP (dbcAMP), on Ca2+ influx triggered by Ca2+ store depletion in the human monocytic cell line, U937. 2. In differentiated cells, the rise in intracellular [Ca2+] following store depletion by thapsigargin (TG) in nominally Ca(2+)-free solution was 94% greater and the [Ca2+]i rise on subsequent re-addition of external Ca2+ (2 mM) was 292% greater than in undifferentiated cells. 3. Under conditions where [Ca2+]i was buffered by BAPTA, TG-induced store depletion failed to activate a detectable inward Ca2+ current in undifferentiated U937 cells. Under identical conditions, store depletion of differentiated U937 cells generated an inwardly rectifying Ca(2+)-selective current which showed no reversal from -140 to +30 mV and was blocked by 1 microM external La3+; characteristics of the calcium release-activated Ca2+ current (ICRAC) identified in other cells. 4. We conclude that U937 cells show a differentiation-dependent upregulation of a store-mediated Ca2+ entry pathway, identified as ICRAC, which is not correlated with the small associated increase in the size of TG-sensitive Ca2+ pools.

IgG-induced Ca2+ oscillations in differentiated U937 cells; a study using laser scanning confocal microscopy and co-loaded fluo-3 and fura-red fluorescent probes.

We have investigated, at the single cell level, intracellular Ca2+ ([Ca2+]i) modulations triggered by the high affinity receptor for IgG, Fc gamma RI, in the monocytic cell line, U937. Cells were co-loaded with the Ca(2+)-sensitive dyes, Fluo-3 and Fura-Red, by incubation with their acetoxymethyl (AM) esters and confocal ratio imaging was used to monitor the [Ca2+]i changes induced by antibody cross-linking of IgG-loaded Fc gamma RI. A single Ca2+ spike was observed in 81% of untreated cells whereas dibutyryl cAMP-induced differentiation into a more macrophage cell type resulted in a sub-population of cells (44%) responding to receptor cross-linking with calcium oscillations. This change in calcium signalling may explain the difference in functional responses triggered by Fc gamma RI in monocytes and macrophages. Analysis of the Fluo-3 and Fura-Red fluorescence, after AM-ester loading, showed that both dyes have similar photobleach rates and intracellular localization allowing compensation for shifts in focal plane, dye photobleaching and non-uniformity of dye loading. In addition, because the binding kinetics of both dyes are equivalent, accurate temporal information can be gained about [Ca2+] changes. There are, however, two major problems with this dual indicator technique. Firstly, loading from AM esters results in considerable variation between cells in the intracellular concentration ratio of the two dyes, making calibration difficult. Secondly, the fluorescence ratio, Fluo-3/Fura-Red, behaves non-linearly at Ca2+ concentrations less than approximately 500 nM and comparison with Fura-2-loaded single cell photometry studies suggests there is considerable amplitude distortion of the signal when the ratios are displayed on a linear scale. These problems may considerably limit the application of Fluo-3/Fura-Red ratiometric measurements.

Fc gamma receptor-mediated phagocytosis requires tyrosine kinase activity and is ligand independent.

Receptors for the invariant chain of immunoglobulins (FcR) define the cellular response to specific antigens. Fc gamma R recognize IgG and so elicit a variety of effector functions including phagocytosis. We are interested in the structural determinants for Fc gamma R-mediated phagocytosis, specifically Fc gamma RI(p135) and Fc gamma RIIa isoforms. The low-affinity receptor, Fc gamma RIIa, is found on macrophages and its cytoplasmic domain contains a tyrosine activation motif which has previously been shown to regulate endocytosis. In contrast, Fc gamma RI has no known signaling motifs, though a functional interaction has recently been demonstrated with the gamma chain of the high-affinity receptor for IgE, Fc epsilon RI. This accessory molecule has a cytoplasmic tyrosine activation motif implicated in signal transduction. Here we demonstrate that although Fc gamma RI transiently expressed on COS-7 cells is able to rosette opsonized SRBC, it cannot phagocytose them. If the cytoplasmic domain of either gamma chain or Fc gamma RIIa replaces that of Fc gamma RI in a chimeric receptor, efficient phagocytosis occurs. This particle ingestion is sensitive to the tyrosine kinase inhibitor genistein. Chimeric receptors where the extracellular domain of either Fc gamma RI or Fc gamma RIIa is replaced with that of CD2, a T cell antigen, indicate that Fc gamma R-mediated phagocytosis is ligand independent. We conclude that phagocytosis is dependent upon close particle apposition, tyrosine kinase activity, and that the process is ligand independent.