Decannulation of patients on peripheral veno-arterial extracorporeal membrane oxygenation (VA ECMO) typically involves femoral arterial cutdown and surgical arteriotomy closure in an operating room under general anesthesia. However, this requires exposure of the still-fragile ECMO patient to the risks of transport from the intensive care unit (ICU), the depressive cardiovascular effects of general anesthesia, and often of re-induction and intubation of a patient who passed their ECMO wean while awake and extubated. We describe a case series in which a novel technique for percutaneous closure of peripheral, percutaneously placed veno-arterial ECMO sites has allowed for safe and reliable closure is achieved in awake, extubated patients, at bedside in the ICU. Common femoral arterial (CFA) sites are closed by accessing the arterial cannula with an 18ga needle, introducing a wire into the CFA over which the arterial cannula is removed and the site subsequently closed with a Teleflex MANTA Closure Device and the superficial femoral artery reperfusion site with a Mynx Vascular Closure Device. In our ten-patient series, complications were minimal, with none developing limb ischemia requiring procedural intervention, failed hemostasis, conversion to cutdown, or requiring intubation or conversion to general anesthesia. In one case, wire access to the artery was lost prematurely but hemostasis was maintained with manual pressure and no further intervention was required. Percutaneous bedside closure of peripheral VA ECMO sites appears feasible and safe. Further study would help hone technique as well as elucidate complication rates and cost saving compared with traditional techniques.
Epidemiological evidence suggests that post-menopausal women are more susceptible to HIV infection following sexual intercourse than are younger cohorts for reasons that remain unclear. Here, we evaluated how menopause-associated changes in CD4 + T cell numbers and subsets as well as HIV coreceptor expression, particularly CCR5, in the endometrium (EM), endocervix (CX), and ectocervix (ECX) may alter HIV infection susceptibility. Using a tissue-specific mixed cell infection model, we demonstrate that while no changes in CD14 + macrophage infection susceptibility were observed, CD4 + T cell HIV-1 infection frequency increases following menopause in the EM, but not CX nor ECX. Unexpectedly, the CD4 + T cell expression of two known correlates of HIV infection susceptibly, CCR5 and integrin-α4β7, increased following menopause across all three tissues despite only being associated with increased infection frequency in EM derived CD4 + T cells. After controlling for changes in the expression of either receptor, both CCR5 and α4β7 expressing CD4 + T cells isolated from the EM of post-menopausal women remained more susceptible to HIV-1 infection than those isolated from pre-menopausal women. Shifts in T helper subset composition, including increases in Th1 frequency and decreases in Th17 and Treg frequency were also observed in the EM only following menopause, but did not correlate with increased infection frequency. Treatment of EM derived CD4 + T cells with 17β-estradiol (E 2 ) prior to viral infection, reduced infection frequency independent of changes in either CCR5 or α4β7 expression frequency. Our results demonstrate that the susceptibility of EM derived CD4 + T cells to HIV-1 infection increases post menopause but is unlikely to be driven by increased expression frequency of either CCR5 or integrin-α4β7. These findings contribute to our understanding of how advanced age alters HIV infection risk which will become increasingly important as the human population continues to age.
In this work, we have investigated the role of bacterial motility with regard to antibiotic-tolerant bacterial aggregate formation. Previous work has convincingly demonstrated that P. aeruginosa flagellar motility promotes the formation of surface-adhered biofilms in many systems. In contrast, aggregate formation by P. aeruginosa was observed for nonmotile but not for motile cells in the presence of an exogenous scaffold. Here, we demonstrate that both wild-type P. aeruginosa and mutants that genetically lack motility spontaneously form antibiotic-tolerant aggregates in the absence of an exogenously added scaffold. Additionally, we also demonstrate that wild-type (WT) and nonmotile P. aeruginosa bacteria can coaggregate, shedding light on potential physiological interactions and heterogeneity of aggregates.
Pathogenic bacteria that establish chronic infections in immunocompromised patients frequently undergo adaptation or selection for traits that are advantageous for their growth and survival. Clinical isolates of Pseudomonas aeruginosa, a Gram-negative, opportunistic bacterial pathogen, exhibit a temporal transition from a motile to a nonmotile phenotype through loss of flagellar motility during the course of chronic infection. This progressive loss of motility is associated with increased resistance to both antibiotic and immune clearance. We have previously shown that loss of bacterial motility enables P. aeruginosa to evade phagocytic clearance both in vitro and in vivo and fails to activate the phosphatidylinositol 3-kinase (PI3K)/Akt-dependent phagocytic pathway. Therefore, we tested the hypothesis that clearance of phagocytosis-resistant bacteria could be induced by exogenously pretreating innate immune cells with the Akt-activating molecule phosphatidylinositol-(3,4,5)-trisphosphate (PIP3). Here, we demonstrate that PIP3 induces the uptake of nonmotile P. aeruginosa by primary human neutrophils >25-fold, and this effect is phenocopied with the use of murine phagocytes. However, surprisingly, mechanistic studies revealed that the induction of phagocytosis by PIP3 occurs because polyphosphoinositides promote bacterial binding by the phagocytes rather than bypassing the requirement for PI3K. Moreover, this induction was selective since the uptake of other nonmotile Gram-negative, but not Gram-positive, bacteria can also be induced by PIP3 Since there is currently no treatment that effectively eradicates chronic P. aeruginosa infections, these findings provide novel insights into a potential methodology by which to induce clearance of nonmotile pathogenic bacteria and into the endogenous determinants of phagocytic recognition of P. aeruginosa.
Estradiol (E 2 ) and progesterone (P) have potent effects on immune function in the human uterine endometrium which is essential for creating an environment conducive for successful reproduction. Type III/lambda (λ) interferons (IFN) are implicated in immune defense of the placenta against viral pathogens, which occurs against the backdrop of high E 2 and P levels. However, the effect of E 2 and P in modulating the expression and function of IFNλ1 in the non-pregnant human uterine endometrium is unknown. We generated purified in vitro cultures of human uterine epithelial cells and stromal fibroblast cells recovered from hysterectomy specimens. Poly (I:C), a viral dsRNA mimic, potently increased secretion of IFNλ1 by both epithelial cells and fibroblasts. The secretion of IFNλ1 by epithelial cells significantly increased with increasing age following poly (I:C) stimulation. Stimulation of either cell type with E 2 (5x10 -8 M) or P (1x10 -7 M) had no effect on expression or secretion of IFNλ1 either alone or in the presence of poly (I:C). E 2 suppressed the IFNλ1-induced upregulation of the antiviral IFN-stimulated genes (ISGs) MxA, OAS2 and ISG15 in epithelial cells, but not fibroblasts. Estrogen receptor alpha (ERα) blockade using Raloxifene indicated that E 2 mediated its inhibitory effects on ISG expression via ERα. In contrast to E 2 , P potentiated the upregulation of ISG15 in response to IFNλ1 but had no effect on MxA and OAS2 in epithelial cells. Our results demonstrate that the effects of E 2 and P on IFNλ1-induced ISGs are cell-type specific. E 2 -mediated suppression, and selective P-mediated stimulation, of IFNλ1-induced ISG expression in uterine epithelial cells suggest that the effects of IFNλ1 varies with menstrual cycle stage, pregnancy, and menopausal status. The suppressive effect of E 2 could be a potential mechanism by which ascending pathogens from the lower reproductive tract can infect the pregnant and non-pregnant endometrium.
Abstract Pseudomonas aeruginosa is a Gram-negative bacterium that, as an opportunistic pathogen, substantially contributes to high morbidity and mortality rates in susceptible individuals such as those with cystic fibrosis or neutropenia. Previous studies identified that the downregulation or loss of bacterial flagellar motility, typically observed within chronic infections, enables bacteria to evade interactions with phagocytic cells that would result in phagocytic uptake. Our recent work demonstrated that exogenous addition of a negatively charged lipid, PIP3, induces binding and phagocytosis of non-motile strains of P. aeruginosa. Based on this work, we hypothesized that the engagement of P. aeruginosa by host innate cells, and subsequent phagocytosis, is mediated by motility-dependent interactions with cell-surface polyanions. We now report that endogenous polyanionic N-linked glycans and heparan sulfate mediate bacterial binding of P. aeruginosa by human monocytic cells. These specific cell-surface interactions result in P. aeruginosa phagocytosis, bacterial type 3 secretion system (T3SS)-mediated cellular intoxication and the IL-1β inflammatory response of the host innate immune cells. Concomitantly, inhibition of host cell N-glycan synthesis reduces T3SS-mediated cytotoxicity and the IL-1β response induced by the bacteria. Importantly, the bacterial interactions with the glycans were motility-dependent and could be recapitulated with purified, immobilized glycans. Therefore, this work describes novel interactions of P. aeruginosa with specific phagocyte cell-surface glycans that modulate relevant host innate immune responses to the bacteria, including phagocytosis, inflammation and cytotoxicity.
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