Support of human cord blood progenitor cells on human stromal cell lines transformed by SV40 large T antigen under the influence of an inducible (metallothionein) promoter

We describe the development of a human bone marrow (BM) culture system which allows study of the interaction of stromal cell lines (SCL) and highly purified hematopoietic progenitor cells. Normal BM stromal cells were electroporated with a plasmid containing the simian virus 40 (SV40) large T antigen (SV40 T Ag) under the control of a synthetic metallothionein promoter (MT4); this construct is designated MT4 SV40 T Ag. SCL in which the rate of proliferation could be controlled by altering the zinc (Zn) concentration were characterized, demonstrating that the SCL were heterogeneous with respect to G-CSF and GM-CSF production. Suppression of SCL proliferation on removal of Zn made it possible to use these lines in coculture with purified CD34+ progenitor cells from umbilical cord blood. The ability to control proliferation of SCL has allowed us to maintain the survival and expansion of colony- forming cells in culture for up to 2 months. These lines have enabled us to test for stromal cell characteristics at a clonal level and provided us with a tool to analyze the events leading to lineage commitment and hematopoietic differentiation, as demonstrated by suppression of hematopoiesis by an antibody directed against the c-kit molecule.     

Involvement of cysteinyl leukotrienes in angiotensin II-induced contraction in isolated aortas from transgenic (mRen-2)27 rats

OBJECTIVES : We have previously reported that 5-lipoxygenase-derived products, and particularly the cysteinyl leukotrienes (CysLTs), were involved in angiotensin II (Ang II)-induced contractions in isolated aortas from spontaneously hypertensive rats. DESIGN : The aim of this study was to assess the role of CysLTs in the vascular response to Ang II in an Ang II-dependent model of hypertension, the (mRen-2)27 transgenic rats (TGs). METHODS : Intact aortic rings from TG and normotensive Sprague-Dawley rats (SDs) were suspended in organ chambers for isometric tension development in response to Ang II. In addition, the release of CysLTs in response to Ang II (0.3 micromol/l) was measured by enzyme immunoassay. RESULTS : In isolated aortas from TG rats, pretreatment with the 5-lipoxygenase inhibitor (AA861, 10 micromol/l) or the CysLT1 receptor antagonist (MK571, 1 micromol/l) significantly (P < 0.05) reduced Ang II-induced contractions by 52 and 42%, respectively. In addition, Ang II induced a 2.6-fold increase in CysLT release (pg/mg dry weight tissue: 58.3 +/- 17.9 (Ang II, n = 7) versus 22.5 +/- 5.9 (basal, n = 7) P < 0.05), which was inhibited by the AT1 receptor antagonist losartan (1 micromol/l). In contrast, in aortas from SD rats, pretreatment with AA861 or MK571 did not alter Ang II-induced contraction and CysLT production remained unchanged after exposure to Ang II. CONCLUSION : These data suggest that CysLTs are involved in the contractile responses to Ang II in isolated aortas from TG but not from SD rats.     

A small region of the beta-adrenergic receptor is selectively involved in its rapid regulation.

Plasma membrane receptors that couple to guanine nucleotide-binding regulatory proteins (G proteins) undergo a variety of rapid (minutes) and longer term (hours) regulatory processes induced by ligands. For the beta 2-adrenergic receptor (beta 2AR), the rapid processes include functional desensitization, mediated by phosphorylation of the receptor by the cAMP-dependent protein kinase and the beta-adrenergic receptor kinase, as well as a loss of hydrophilic ligand binding proposed to represent sequestration of receptors into a cellular compartment distinct from the plasma membrane. The slower processes include beta 2AR down-regulation (i.e., a decrease in the total cellular complement of receptors). It is not yet known whether beta 2AR phosphorylation and/or sequestration are prerequisites for down-regulation of the receptor. Like other G protein-coupled receptors, the beta 2AR molecule spans the plasma membrane seven times, and the cytoplasmic carboxyl-terminal domain has been proposed to contain molecular determinants for each of these regulatory processes. We replaced four serine and threonine residues located within a 10-amino acid segment of this domain of beta 2AR and thereby prevented agonist-promoted phosphorylation, sequestration, and rapid desensitization of the adenylyl cyclase response. In contrast, these mutations did not affect functional coupling to the stimulatory G protein Gs or long-term down-regulation. These findings thus define a small, hitherto unappreciated region of the receptor molecule that may selectively subserve its rapid regulation. In addition, with the demonstration that beta 2AR does not have to be phosphorylated or sequestered in order to enter the down-regulation pathway, the results suggest that the classical receptor endocytosis model may not be appropriate for beta 2AR regulation.     

Role for G protein-coupled receptor kinase in agonist-specific regulation of μ-opioid receptor responsiveness

The G protein-coupled μ-opioid receptor (μOR) mediates the physiological effects of endogenous opioid peptides as well as the structurally distinct opioid alkaloids morphine and etorphine. An intriguing feature of μOR signaling is the differential receptor trafficking and desensitization properties following activation by distinct agonists, which have been proposed as possible mechanisms related to opioid tolerance. Here we report that the ability of distinct opioid agonists to differentially regulate μOR internalization and desensitization is related to their ability to promote G protein-coupled receptor kinase (GRK)-dependent phosphorylation of the μOR. Although both etorphine and morphine effectively activate the μOR, only etorphine elicits robust μOR phosphorylation followed by plasma membrane translocation of β-arrestin and dynamin-dependent receptor internalization. In contrast, corresponding to its inability to cause μOR internalization, morphine is unable to either elicit μOR phosphorylation or stimulate β-arrestin translocation. However, upon the overexpression of GRK2, morphine gains the capacity to induce μOR phosphorylation, accompanied by the rescue of β-arrestin translocation and receptor sequestration. Moreover, overexpression of GRK2 also leads to an attenuation of morphine-mediated inhibition of adenylyl cyclase. These findings point to the existence of marked differences in the ability of different opioid agonists to promote μOR phosphorylation by GRK. These differences may provide the molecular basis underlying the different analgesic properties of opioid agonists and contribute to the distinct ability of various opioids to induce drug tolerance.     

Pheochromocytoma in multiple endocrine neoplasia type 2: a prospective study

OBJECTIVE: The aim of this prospective study is to update our knowledge of the chronology of pheochromocytoma occurrence in multiple endocrine neoplasia type 2 (MEN 2), and to better manage MEN 2 patients after the genetic diagnosis. DESIGN: Eighty-seven non-index gene carrier MEN 2 patients were included in this prospective study: 84 patients with MEN 2A (from 52 families) and 3 with MEN 2B (from 3 families). METHODS: Medullary thyroid carcinoma (MTC) was diagnosed by measuring plasma calcitonin in basal conditions or after pentagastrin stimulation. The search for pheochromocytoma consisted of clinical evaluation, 24 h determination of urinary catecholamines and adrenal imaging. The mean age at genetic diagnosis of MEN 2 was 14.0+/-7.0 years, the mean duration for the follow-up was 7.6+/-2.8 years. RESULTS: All 87 patients had a MTC detected at the same time as the genetic diagnosis was made. Urinary catecholamine measurements led to the diagnosis of pheochromocytoma and a combination of imaging techniques enabled the correct localization of both unilateral or bilateral adrenal involvement. Pheochromocytoma was detected simultaneously with MTC in only seven patients, and seven others were detected throughout the follow-up. Of the 14 patients with pheochromocytoma, 11 had bilateral involvement: nine were initially bilateral and two became so during follow-up. CONCLUSION: This study demonstrates that in MEN 2, MTC is the lesion which appears earliest. Pheochromocytoma develops later during the evolution of the disease, and necessitates regular clinical and biological monitoring throughout follow-up. Determination of urinary and/or plasma catecholamines and metanephrines should be performed to detect pheochromocytoma. Imaging techniques lead to the detection of both unilateral and bilateral pheochromocytoma, thus making video-assisted laparoscopic adrenalectomy possible.     

The Relationship Between Parental Stress and Postpartum Depression Among Adolescent Mothers Enrolled in a Randomized Controlled Prevention Trial

Given the high co-occurrence of depression and parental stress among adolescent mothers, we evaluated the relationship between parental stress and postpartum depression among primiparous adolescent mothers. We conducted an observational analysis among a cohort of 106 adolescent mothers at 289 postpartum visits who were enrolled in a randomized controlled trial to prevent postpartum depression. Parental stress was measured using the Parenting Stress Index, short form. The Structured Clinical Interview for DSM-IV Childhood Diagnoses was administered to assess for postpartum depression; subthreshold depression was assessed using the Children’s Depression Rating Scale, revised version. Generalized estimating equations were utilized to assess the relationship of parental stress on postpartum depression during the first 6 months postpartum. We present adjusted odds ratios (AOR) controlling for study arm, age, born in the United States, prior history of depression, and number of study visits. The median age was 16 years, 53 % were Latina, and 16 % reported a past history of depression. Nineteen adolescents (19 %) were diagnosed with postpartum depression and 25 % experienced high levels of parental stress through 6 months postpartum. Adolescent mothers who reported higher levels of parental stress were at significantly increased risk for postpartum depression [AOR 1.06 (95 % CI 1.04–1.09); p < 0.0001]. High levels of parental stress predicted subsequent postpartum depression when assessing parental stress at visits prior to a depression diagnosis to determine whether we could establish a temporal association [AOR 1.06 (95 % CI 1.02–1.09); p < 0.01]. Parental stress was also a risk factor for subthreshold depression [AOR 1.04 (95 % CI 1.01–1.07); p < 0.01]. Parental stress was a significant risk factor for developing both postpartum depression as well as subthreshold depression among adolescent mothers. Interventions that target a reduction in parental stress may lead to less depression severity among primiparous adolescent mothers.     

Tractostorm 2: Optimizing tractography dissection reproducibility with segmentation protocol dissemination

The segmentation of brain structures is a key component of many neuroimaging studies. Consistent anatomical definitions are crucial to ensure consensus on the position and shape of brain structures, but segmentations are prone to variation in their interpretation and execution. White‐matter (WM) pathways are global structures of the brain defined by local landmarks, which leads to anatomical definitions being difficult to convey, learn, or teach. Moreover, the complex shape of WM pathways and their representation using tractography (streamlines) make the design and evaluation of dissection protocols difficult and time‐consuming. The first iteration of Tractostorm quantified the variability of a pyramidal tract dissection protocol and compared results between experts in neuroanatomy and nonexperts. Despite virtual dissection being used for decades, in‐depth investigations of how learning or practicing such protocols impact dissection results are nonexistent. To begin to fill the gap, we evaluate an online educational tractography course and investigate the impact learning and practicing a dissection protocol has on interrater (groupwise) reproducibility. To generate the required data to quantify reproducibility across raters and time, 20 independent raters performed dissections of three bundles of interest on five Human Connectome Project subjects, each with four timepoints. Our investigation shows that the dissection protocol in conjunction with an online course achieves a high level of reproducibility (between 0.85 and 0.90 for the voxel‐based Dice score) for the three bundles of interest and remains stable over time (repetition of the protocol). Suggesting that once raters are familiar with the software and tasks at hand, their interpretation and execution at the group level do not drastically vary. When compared to previous work that used a different method of communication for the protocol, our results show that incorporating a virtual educational session increased reproducibility. Insights from this work may be used to improve the future design of WM pathway dissection protocols and to further inform neuroanatomical definitions.     

Platelet-Derived MRP-14 Induces Monocyte Activation in Patients With Symptomatic Peripheral Artery Disease

Background

Peripheral artery disease (PAD), a diffuse manifestation of atherothrombosis, is a major cardiovascular threat. Although platelets are primary mediators of atherothrombosis, their role in the pathogenesis of PAD remains unclear.

Emergency air evacuation of patients with acute respiratory failure due to SARS-CoV-2 from Mayotte to Reunion Island

In February 2021, an explosion of cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pneumonia overwhelmed the only hospital in Mayotte. To report a case series of patients with acute respiratory failure (ARF) due to SARS-CoV-2 who were evacuated by air from Mayotte to Reunion Island.This retrospective observational study evaluated all consecutive patients with ARF due to SARS-CoV-2 who were evacuated by air from Mayotte Hospital to the intensive care unit (ICU) of Félix Guyon University Hospital in Reunion Island between February 2, and March 5, 2021.A total of 43 patients with SARS-CoV-2 pneumonia were evacuated by air, for a total flight time of 2 hours and a total travel time of 6 hours. Of these, 38 patients (88.4%) with a median age of 55 (46–65) years presented with ARF and were hospitalized in our ICU. Fifteen patients were screened for the SARS-CoV-2 501Y.V2 variant, all of whom tested positive. Thirteen patients (34.2%) developed an episode of severe hypoxemia during air transport, and the median paO₂/FiO₂ ratio was lower on ICU admission (140 [102–192] mmHg) than on departure (165 [150–200], P = .022). Factors associated with severe hypoxemia during air transport was lack of treatment with curare (P = .012) and lack of invasive mechanical ventilation (P = .003). Nine patients (23.7%) received veno-venous extracorporeal membrane oxygenation support in our ICU. Seven deaths (18.4%) occurred in hospital.Emergency air evacuation of patients with ARF due to SARS-CoV-2 was associated with severe hypoxemia but remained feasible. In cases of ARF due to SARS-CoV-2 requiring emergency air evacuation, sedated patients receiving invasive mechanical ventilation and curare should be prioritized over nonintubated patients. It is noteworthy that patients with SARS-CoV-2 pneumonia related to the 501Y.V2 variant were very severe despite their young age.