DC-SIGN(+) Macrophages Control the Induction of Transplantation Tolerance

Tissue effector cells of the monocyte lineage can differentiate into different cell types with specific cell function depending on their environment. The phenotype, developmental requirements, and functional mechanisms of immune protective macrophages that mediate the induction of transplantation tolerance remain elusive. Here, we demonstrate that costimulatory blockade favored accumulation of DC-SIGN-expressing macrophages that inhibited CD8(+) T cell immunity and promoted CD4(+)Foxp3(+) Treg cell expansion in numbers. Mechanistically, that simultaneous DC-SIGN engagement by fucosylated ligands and TLR4 signaling was required for production of immunoregulatory IL-10 associated with prolonged allograft survival. Deletion of DC-SIGN-expressing macrophages in vivo, interfering with their CSF1-dependent development, or preventing the DC-SIGN signaling pathway abrogated tolerance. Together, the results provide new insights into the tolerogenic effects of costimulatory blockade and identify DC-SIGN(+) suppressive macrophages as crucial mediators of immunological tolerance with the concomitant therapeutic implications in the clinic.This work was supported by the COST Action BM1305: Action to Focus and Accelerate Cell Tolerogenic Therapies (A FACTT), the Mount Sinai Recanati/Miller Transplantation Institute developmental funds, AST/Pfizer Basic Science Faculty Development Grant, Ministerio de Educacióny Ciencia SAF2010-15062, SAF2013-48834-R, and Fundación Mutua Madrileñ a grants to J.O. A portion of this work appears as part of the doctoral thesis of P.C.S

The genomic landscape of balanced cytogenetic abnormalities associated with human congenital anomalies

Despite the clinical significance of balanced chromosomal abnormalities (BCAs), their characterization has largely been restricted to cytogenetic resolution. We explored the landscape of BCAs at nucleotide resolution in 273 subjects with a spectrum of congenital anomalies. Whole-genome sequencing revised 93% of karyotypes and demonstrated complexity that was cryptic to karyotyping in 21% of BCAs, highlighting the limitations of conventional cytogenetic approaches. At least 33.9% of BCAs resulted in gene disruption that likely contributed to the developmental phenotype, 5.2% were associated with pathogenic genomic imbalances, and 7.3% disrupted topologically associated domains (TADs) encompassing known syndromic loci. Remarkably, BCA breakpoints in eight subjects altered a single TAD encompassing MEF2C, a known driver of 5q14.3 microdeletion syndrome, resulting in decreased MEF2C expression. We propose that sequence-level resolution dramatically improves prediction of clinical outcomes for balanced rearrangements and provides insight into new pathogenic mechanisms, such as altered regulation due to changes in chromosome topology

Control of resting bronchial hemodynamics in the awake dog

In the resting awake dog a continuous-wave Doppler flow transducer on the right bronchoesophageal artery inscribes a sharp early systolic spike and low flow in late systole and throughout diastole, indicating a highly resistive bed. An analysis of autonomic factors using intravenous, cumulative, and randomly applied cholinoceptor, beta 1- and beta 2-adrenoceptor, and alpha 1- and alpha 2-adrenoceptor antagonists indicates that the low vascular conductance is due to cholinoceptor and alpha 1- and alpha 2-adrenoceptor effects in a ratio 3.6:1. No beta-adrenoceptor tone is present. Sighing behavior invokes a transient (< 2 s) fall in intrapleural pressure (and thus rise in bronchovascular transmural pressure) of 10-30 mmHg, which is followed by a two- to threefold increase over 30 s in bronchial flow and conductance, an effect simulated in 50% of dogs when bronchovascular transmural pressure is acutely raised and maintained over 40-60 s by inflating an intra-aortic balloon distal to the origin of the bronchial artery. Autonomic blockade has no effect on bronchovascular dilatation evoked either by sighing or by balloon inflation. It is concluded that, in the resting bronchial circulation, there exists strong cholinoceptor and alpha-adrenoceptor-based vasoconstrictor activity which can be overpowered by strong nonadrenergic noncholinergic local vasodilator reflexes evoked by sudden changes in intrathoracic transmural pressure possibly acting on stretch-sensitive sensory nerve endings containing substance P, calcitonin gene-related peptide, and neurokinins. The tonic vasoconstrictor but not the sigh-evoked vasodilator effects are sensitive to pentobarbital sodium anesthesia

Cardioselective inhibition of sympathetic control by atenolol in man does not evoke fatigue: a double-blind study

Cardioselective sympathetic blockade is useful in clinical management but the side-effect of fatigue (enhanced tiredness during exercise) suggests that physiological compensations are inadequate. Pilot studies confirmed that the effects of exercise on cardiac output (CO; impedance cardiography), serum lactate (SL) and rating of perceived exertion (RPE) were reproducible within subjects and between weeks over 4 weekly tests. In 6 untrained normotensive men (age 41.1±2.13yr) the effects of 2 doses 24hr apart of either atenolol 50 mg (A), atenolol 50mg plus chlorthalidone 12.5mg (AC), placebo 1 or placebo 2, were studied at rest and during incremental exercise to maximum on 4 occasions in each subject 1 week apart using a double blind, randomized protocol. The use of A was marked by lassitude (a feeling of tiredness at rest). On average both A and A+C each lowered resting and exercise heart rate, arterial pressure and CO to a similar degree. Nevertheless, the external work performed and the exercise related rise in RPE and SL was similar irrespective of the drug used as was the level to which total peripheral resistance fell. Thus selective cardiac sympatholysis does not result in fatigue or its potential metabolic causes at rest or over the full range of effort. Lassitude is possibly due to minimal penetration of the blood brain barrier by atenolol

Dynamic range of evoked cholinergic and non-cholinergic non-adrenergic vasodilatation in bronchoesphageal circulation in the awake dog

The functional characteristics and control factors of the bronchoesophageal circulation under natural conditions are unknown. In 8 awake dogs 2-3 weeks after mounting a 3mm i.d. Doppler flow probe on the right bronchoesophageal artery under halothane-oxygen anaesthesia, the bronchial circulation is a low-flow, high resistance bed under resting thermoneutral conditions (mean bronchovascular flow (Qbr) = 0.27ml·s⁻¹, range 0.03-0.75, n=8; mean bronchovascular conductance (Cbr) = 8.5x10⁻⁴ml·s⁻¹·mmHg⁻¹, range 2x10⁻⁴21x10⁻⁴, n=5). Sequential autonomic blockade (i.v. methscopolamine 270 μg·kg⁻¹; propranolol 1 mg·kg⁻¹; phentolamine 4mg followed by 0.15−0·4 mg·min⁻¹ infusion) reveals significant resting α-adrenoceptor vasoconstrictor tone and suggest the presence of resting cholinoceptor vasoconstrictor tone. Notwithstanding acetylcholine (0.063-2·0 μg·kg⁻¹, i.v. bolus) evokes transient increased in Qbr (2-7 times; ED₅₀ 0.68 μg·kg⁻¹) and in Cbr (2-8 times; ED₅₀ 0.46 μg·kg⁻¹). Rapid (<ls) increased in Qbr and Cbr were evoked with sighs followed by apnoea and by raising aortic pressure (Pa) ~ 20mmHg (inflation of Fogarty balloon in thoracic aorta via femoral arteriotomy, local lidocaine anaesthesia). Autonomic blockade does not diminish the Qbr and Cbr responses, and responses to balloon inflation persist while Pa remains elevated. Thus noncholinergic nonadrenergic mechanisms play a substantial role together with conventional autonomic mechanisms in regulating the normal bronchoesophageal circulation

Dynamic control of the bronchial circulation in the conscious dog: preliminary data on the role of alpha and beta adrenoceptors, and of cholinoceptors

Due to a unique vascular network, bronchial blood flow may be determined by the opening of anastomotic channels during perfusion pressure changes as well as by neurohumoral mechanisms. For example, previous studies from this laboratory have shown that a rise in aortic pressure, or a sigh followed by apnoea, result in dramatic rises (< 1s time-constant) in bronchial flow and conductance, which are not diminished by conventional autonomic blockade. These data suggest that unusual mechanical effects may indeed be involved, or that unknown reflex pathways are active. In order to define further the potential neurohumoral factors regulating bronchial flow, the present studies were designed to examine the role of a- and a-adrenoceptor, and cholinoceptor control. While the presence of such receptor mechanisms in the bronchial bed are likely the data were derived from indirect techniques for estimating airway vascular activity in anaesthetized, open-chested dogs. By contrast, in this study the continuous-wave Doppler flowmeter was used to examine the dynamic effects of these receptor control systems on bronchial flow in the conscious dog

Nanogap Dielectric Spectroscopy for Aptamer-Based Protein Detection

Among the various label-free methods for monitoring biomolecular interactions, capacitive sensors stand out due to their simple instrumentation and compatibility with multiplex formats. However, electrode polarization due to ion gradient formation and noise from solution conductance limited early dielectric spectroscopic measurements to high frequencies only, which in turn limited their sensitivity to biomolecular interactions, as the applied excitation signals were too fast for the charged macromolecules to respond. To minimize electrode polarization effects, capacitive sensors with 20 nm electrode separation were fabricated using silicon dioxide sacrificial layer techniques. The nanoscale separation of the capacitive electrodes in the sensor results in an enhanced overlapping of electrical double layers, and apparently a more ordered “ice-like” water structure. Such effects in turn reduce low frequency contributions from bulk sample resistance and from electrode polarization, and thus markedly enhance sensitivity toward biomolecular interactions. Using these nanogap capacitive sensors, highly sensitive, label-free aptamer-based detection of protein molecules is achieved