Treating patients as persons : a capabilities approach to support delivery of person-centered care

Peer reviewedPublisher PD

Coxiella burnetii Phagocytosis Is Regulated by GTPases of the Rho Family and the RhoA Effectors mDia1 and ROCK

The GTPases belonging to the Rho family control the actin cytoskeleton rearrangements needed for particle internalization during phagocytosis. ROCK and mDia1 are downstream effectors of RhoA, a GTPase involved in that process. Coxiella burnetii, the etiologic agent of Q fever, is internalized by the host´s cells in an actin-dependent manner. Nevertheless, the molecular mechanism involved in this process has been poorly characterized. This work analyzes the role of different GTPases of the Rho family and some downstream effectors in the internalization of C. burnetii by phagocytic and non-phagocytic cells. The internalization of C. burnetii into HeLa and RAW cells was significantly inhibited when the cells were treated with Clostridium difficile Toxin B which irreversibly inactivates members of the Rho family. In addition, the internalization was reduced in HeLa cells that overexpressed the dominant negative mutants of RhoA, Rac1 or Cdc42 or that were knocked down for the Rho GTPases. The pharmacological inhibition or the knocking down of ROCK diminished bacterium internalization. Moreover, C. burnetii was less efficiently internalized in HeLa cells overexpressing mDia1-N1, a dominant negative mutant of mDia1, while the overexpression of the constitutively active mutant mDia1-ΔN3 increased bacteria uptake. Interestingly, when HeLa and RAW cells were infected, RhoA, Rac1 and mDia1 were recruited to membrane cell fractions. Our results suggest that the GTPases of the Rho family play an important role in C. burnetii phagocytosis in both HeLa and RAW cells. Additionally, we present evidence that ROCK and mDia1, which are downstream effectors of RhoA, are involved in that processFil: Salinas Ojeda, Romina Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Ortiz Flores, Rodolfo Matias. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Distel, Jesús Sebastián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Aguilera, Milton Osmar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Colombo, Maria Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Beron, Walter. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentin

Tn1546 is part of a larger plasmid-encoded genetic unit horizontally disseminated among clonal Enterococcus faecium lineages

o determine the genetic composition of the first VanA-type plasmid (pIP816) reported, which was isolated from a clinical Enterococcus faecium (BM4147) strain in France in 1986, and to reveal the genetic units responsible for the dissemination of the vanA gene cluster by comparisons with current, published and additionally generated vanA-spanning plasmid sequences obtained from a heterogeneous E. faecium strain collection (n = 28).Plasmid sequences were produced by shotgun sequencing using ABI dye chemistry and primer walking, and were subsequently annotated. Comparative sequence analysis of the vanA region was done with published plasmids, with a partial vanA plasmid (pVEF4) reported here and to >140 kb of sequence obtained from a collection of vanA-harbouring plasmid fragments. Bioinformatic analyses revealed that pIP816 from 1986 and contemporary vanA plasmids shared a conserved genetic fragment of 25 kb, spanning the 10.85 kb vanA cluster encoded by Tn1546, and that the larger unit is present in both clinical and animal complexes of E. faecium. A new group II intron in pVEF4 was characterized. Comparative DNA analyses suggest that Tn1546 disseminates in and between clonal complexes of E. faecium as part of a larger genetic unit, possibly as a composite transposon flanked by IS1216 elements

The Escherichia coli effector EspJ blocks Src kinase activity via amidation and ADP ribosylation

J.C.Y. was funded by an MRC PhD studentship. D.J.B. is supported by a London Research Institute, Cancer Research UK Postdoctoral Fellowship award and M.W. is supported by Cancer Research UK. K.A. was supported by the Deutsche Forschungsgemeinschaft (AK 6/22-1 and AK 6/22-2) and the Center for Biological Signaling Studies in Freiburg (Germany). This work was supported by grants from the Wellcome Trust to G.F. and S.J.M

A Conserved Role for SNX9-Family Members in the Regulation of Phagosome Maturation during Engulfment of Apoptotic Cells

Clearance of apoptotic cells is of key importance during development, tissue homeostasis and wound healing in multi-cellular animals. Genetic studies in the nematode Caenorhabditis elegans have identified a set of genes involved in the early steps of cell clearance, in particular the recognition and internalization of apoptotic cells. A pathway that orchestrates the maturation of phagosomes containing ingested apoptotic cells in the worm has recently been described. However, many steps in this pathway remain elusive. Here we show that the C. elegans SNX9-family member LST-4 (lateral signaling target) and its closest mammalian orthologue SNX33 play an evolutionary conserved role during apoptotic cell corpse clearance. In lst-4 deficient worms, internalized apoptotic cells accumulated within non-acidified, DYN-1-positive but RAB-5-negative phagosomes. Genetically, we show that LST-4 functions at the same step as DYN-1 during corpse removal, upstream of the GTPase RAB-5. We further show that mammalian SNX33 rescue C. elegans lst-4 mutants and that overexpression of truncated SNX33 fragments interfered with phagosome maturation in a mammalian cell system. Taken together, our genetic and cell biological analyses suggest that LST-4 is recruited through a combined activity of DYN-1 and VPS-34 to the early phagosome membrane, where it cooperates with DYN-1 to promote recruitment/retention of RAB-5 on the early phagosomal membrane during cell corpse clearance. The functional conservation between LST-4 and SNX33 indicate that these early steps of apoptotic phagosome maturation are likely conserved through evolution

Characterisation of Innate Fungal Recognition in the Lung

The innate recognition of fungi by leukocytes is mediated by pattern recognition receptors (PRR), such as Dectin-1, and is thought to occur at the cell surface triggering intracellular signalling cascades which lead to the induction of protective host responses. In the lung, this recognition is aided by surfactant which also serves to maintain the balance between inflammation and pulmonary function, although the underlying mechanisms are unknown. Here we have explored pulmonary innate recognition of a variety of fungal particles, including zymosan, Candida albicans and Aspergillus fumigatus, and demonstrate that opsonisation with surfactant components can limit inflammation by reducing host-cell fungal interactions. However, we found that this opsonisation does not contribute directly to innate fungal recognition and that this process is mediated through non-opsonic PRRs, including Dectin-1. Moreover, we found that pulmonary inflammatory responses to resting Aspergillus conidia were initiated by these PRRs in acidified phagolysosomes, following the uptake of fungal particles by leukocytes. Our data therefore provides crucial new insights into the mechanisms by which surfactant can maintain pulmonary function in the face of microbial challenge, and defines the phagolysosome as a novel intracellular compartment involved in the innate sensing of extracellular pathogens in the lung

Immune system deregulation in hypertensive patients chronically RAS suppressed developing albuminuria

Albuminuria development in hypertensive patients is an indicator of higher cardiovascular (CV) risk and renal damage. Chronic renin-angiotensin system (RAS) suppression facilitates blood pressure control but it does not prevent from albuminuria development. We pursued the identification of protein indicators in urine behind albuminuria development in hypertensive patients under RAS suppression. Urine was collected from 100 patients classified in three groups according to albuminuria development: (a) patients with persistent normoalbuminuria; (b) patients developing de novo albuminuria; (c) patients with maintained albuminuria. Quantitative analysis was performed in a first discovery cohort by isobaric labeling methodology. Alterations of proteins of interest were confirmed by target mass spectrometry analysis in an independent cohort. A total of 2416 proteins and 1223 functional categories (coordinated protein responses) were identified. Immune response, adhesion of immune and blood cells, and phagocytosis were found significantly altered in patients with albuminuria compared to normoalbuminuric individuals. The complement system C3 increases, while Annexin A1, CD44, S100A8 and S100A9 proteins showed significant diminishment in their urinary levels when albuminuria is present. This study reveals specific links between immune response and controlled hypertension in patients who develop albuminuria, pointing to potential protein targets for novel and future therapeutic interventions.Sin financiación4.122 JCR (2017) Q1, 12/64 Multidisciplinary Sciences0.809 SJR (2017) Q2, 4/10 OptometryNo data IDR 2017UE

Fcγ Receptor I Alpha Chain (CD64) Expression in Macrophages Is Critical for the Onset of Meningitis by Escherichia coli K1

Neonatal meningitis due to Escherichia coli K1 is a serious illness with unchanged morbidity and mortality rates for the last few decades. The lack of a comprehensive understanding of the mechanisms involved in the development of meningitis contributes to this poor outcome. Here, we demonstrate that depletion of macrophages in newborn mice renders the animals resistant to E. coli K1 induced meningitis. The entry of E. coli K1 into macrophages requires the interaction of outer membrane protein A (OmpA) of E. coli K1 with the alpha chain of Fcγ receptor I (FcγRIa, CD64) for which IgG opsonization is not necessary. Overexpression of full-length but not C-terminal truncated FcγRIa in COS-1 cells permits E. coli K1 to enter the cells. Moreover, OmpA binding to FcγRIa prevents the recruitment of the γ-chain and induces a different pattern of tyrosine phosphorylation of macrophage proteins compared to IgG2a induced phosphorylation. Of note, FcγRIa−/− mice are resistant to E. coli infection due to accelerated clearance of bacteria from circulation, which in turn was the result of increased expression of CR3 on macrophages. Reintroduction of human FcγRIa in mouse FcγRIa−/− macrophages in vitro increased bacterial survival by suppressing the expression of CR3. Adoptive transfer of wild type macrophages into FcγRIa−/− mice restored susceptibility to E. coli infection. Together, these results show that the interaction of FcγRI alpha chain with OmpA plays a key role in the development of neonatal meningitis by E. coli K1

CD14 Signaling Restrains Chronic Inflammation through Induction of p38-MAPK/SOCS-Dependent Tolerance

Current thinking emphasizes the primacy of CD14 in facilitating recognition of microbes by certain TLRs to initiate pro-inflammatory signaling events and the importance of p38-MAPK in augmenting such responses. Herein, this paradigm is challenged by demonstrating that recognition of live Borrelia burgdorferi not only triggers an inflammatory response in the absence of CD14, but one that is, in part, a consequence of altered PI3K/AKT/p38-MAPK signaling and impaired negative regulation of TLR2. CD14 deficiency results in increased localization of PI3K to lipid rafts, hyperphosphorylation of AKT, and reduced activation of p38. Such aberrant signaling leads to decreased negative regulation by SOCS1, SOCS3, and CIS, thereby compromising the induction of tolerance in macrophages and engendering more severe and persistent inflammatory responses to B. burgdorferi. Importantly, these altered signaling events and the higher cytokine production observed can be mimicked through shRNA and pharmacological inhibition of p38 activity in CD14-expressing macrophages. Perturbation of this CD14/p38-MAPK-dependent immune regulation may underlie development of infectious chronic inflammatory syndromes

Fungal dissemination is limited by liver macrophage filtration of the blood

Partial funding for Open Access provided by the UMD Libraries' Open Access Publishing Fund.Fungal dissemination into the bloodstream is a critical step leading to invasive fungal infections. Here, using intravital imaging, we show that Kupffer cells (KCs) in the liver have a prominent function in the capture of circulating Cryptococcus neoformans and Candida albicans, thereby reducing fungal dissemination to target organs. Complement C3 but not C5, and complement receptor CRIg but not CR3, are involved in capture of C. neoformans. Internalization of C. neoformans by KCs is subsequently mediated by multiple receptors, including CR3, CRIg, and scavenger receptors, which work synergistically along with C5aR signaling. Following phagocytosis, the growth of C. neoformans is inhibited by KCs in an IFN-γ independent manner. Thus, the liver filters disseminating fungi from circulation via KCs, providing a mechanistic explanation for the enhanced risk of cryptococcosis among individuals with liver diseases, and suggesting a therapeutic strategy to prevent fungal dissemination through enhancing KC functions