scholarly journals In vivo analysis of Trypanosoma cruzi persistence foci at single cell resolution

2020 ◽  
Author(s):  
Alexander I. Ward ◽  
Michael D. Lewis ◽  
Archie Khan ◽  
Conor J. McCann ◽  
Amanda F. Francisco ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Immune Response ◽  
Smooth Muscle ◽  
Trypanosoma Cruzi ◽  
Drug Development ◽  
Single Cell ◽  
Parasite Burden ◽  
Circular Muscle ◽  
Muscle Layer ◽  
Chronic Infections

ABSTRACTInfections with Trypanosoma cruzi are usually life-long despite generating a strong adaptive immune response. Identifying the sites of parasite persistence is therefore crucial to understand how T. cruzi avoids immune-mediated destruction. However, this is a major technical challenge because the parasite burden during chronic infections is extremely low. Here, we describe an integrated approach involving comprehensive tissue processing, ex vivo imaging, and confocal microscopy, which has allowed us to visualise infected host cells in murine tissue, with exquisite sensitivity. Using bioluminescence-guided tissue sampling, with a detection level of <20 parasites, we show that in the colon, smooth muscle myocytes in the circular muscle layer are the most common infected host cell type. Typically, during chronic infections, the entire colon of a mouse contains only a few hundred parasites, often concentrated in a small number of cells containing >200 parasites, that we term mega-nests. In contrast, during the acute stage, when the total parasite burden is considerably higher and many cells are infected, nests containing >50 parasites are rarely found. In C3H/HeN mice, but not BALB/c, we identified skeletal muscle as a major site of persistence during the chronic stage, with most parasites found in large mega-nests within the muscle fibres. Finally, we report that parasites are also frequently found in the skin during chronic murine infections, often in multiple infection foci. In addition to being a site of parasite persistence, this anatomical reservoir could play an important role in insect-mediated transmission, and have implications for drug development.IMPORTANCETrypanosoma cruzi causes Chagas disease, the most important parasitic infection in Latin America. Major pathologies include severe damage to the heart and digestive tract, although symptoms do not usually appear until decades after infection. Research has been hampered by the complex nature of the disease and technical difficulties in locating the extremely low number of parasites. Here, using highly sensitive imaging technology, we reveal the sites of parasite persistence in experimental mice at single-cell resolution. We show that parasites are frequently located in smooth muscle cells in the circular muscle layer of the colon, and that skeletal muscle cells and the skin can also be important reservoirs. This information provides a framework for investigating how the parasite is able to survive as a life-long infection, despite a vigorous immune response. It also informs drug-development strategies by identifying tissue sites that must be accessed to achieve a curative outcome.

scholarly journals In Vivo Analysis of Trypanosoma cruzi Persistence Foci at Single-Cell Resolution

mBio ◽  
2020 ◽  
Vol 11 (4) ◽  
Author(s):  
Alexander I. Ward ◽  
Michael D. Lewis ◽  
Archie A. Khan ◽  
Conor J. McCann ◽  
Amanda F. Francisco ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Immune Response ◽  
Smooth Muscle ◽  
Trypanosoma Cruzi ◽  
Parasite Burden ◽  
Circular Muscle ◽  
Muscle Layer ◽  
Chronic Infections ◽  
Content Type ◽  
Chronic Stage

ABSTRACT Infections with Trypanosoma cruzi are usually lifelong despite generating a strong adaptive immune response. Identifying the sites of parasite persistence is therefore crucial to understanding how T. cruzi avoids immune-mediated destruction. However, this is a major technical challenge, because the parasite burden during chronic infections is extremely low. Here, we describe an integrated approach involving comprehensive tissue processing, ex vivo imaging, and confocal microscopy, which allowed us to visualize infected host cells in murine tissue with exquisite sensitivity. Using bioluminescence-guided tissue sampling, with a detection level of <20 parasites, we showed that in the colon, smooth muscle myocytes in the circular muscle layer are the most common infected host cell type. Typically, during chronic infections, the entire colon of a mouse contains only a few hundred parasites, often concentrated in a small number of cells each containing >200 parasites, which we term mega-nests. In contrast, during the acute stage, when the total parasite burden is considerably higher and many cells are infected, nests containing >50 parasites are rarely found. In C3H/HeN mice, but not BALB/c mice, we identified skeletal muscle as a major site of persistence during the chronic stage, with most parasites being found in large mega-nests within the muscle fibers. Finally, we report that parasites are also frequently found in the skin during chronic murine infections, often in multiple infection foci. In addition to being a site of parasite persistence, this anatomical reservoir could play an important role in insect-mediated transmission and have implications for drug development. IMPORTANCE Trypanosoma cruzi causes Chagas disease, the most important parasitic infection in Latin America. Major pathologies include severe damage to the heart and digestive tract, although symptoms do not usually appear until decades after infection. Research has been hampered by the complex nature of the disease and technical difficulties in locating the extremely low number of parasites. Here, using highly sensitive imaging technology, we reveal the sites of parasite persistence during chronic-stage infections of experimental mice at single-cell resolution. We show that parasites are frequently located in smooth muscle cells in the circular muscle layer of the colon and that skeletal muscle cells and the skin can also be important reservoirs. This information provides a framework for investigating how the parasite is able to survive as a lifelong infection, despite a vigorous immune response. It also informs drug development strategies by identifying tissue sites that must be accessed to achieve a curative outcome.

Myosin phosphorylation and contraction of feline esophageal smooth muscle

1985 ◽  
Vol 249 (1) ◽  
pp. C9-C14 ◽  
Author(s):  
N. W. Weisbrodt ◽  
R. A. Murphy
Keyword(s):  
Smooth Muscle ◽  
Mechanical Activation ◽  
Smooth Muscles ◽  
Circular Muscle ◽  
Electrical Field Stimulation ◽  
Muscle Layer ◽  
Field Stimulation ◽  
Shortening Velocity ◽  
Myosin Phosphorylation ◽  
Isotonic Shortening

We tested the hypothesis that phosphorylation of the 20,000-Da light chain of myosin (LC 20) is related to mechanical activation of esophageal smooth muscle. Circular muscle layer strips of cat esophagus were taken from the lower esophageal sphincter (LES) and the distal esophageal body (EB). The LES strips developed tone spontaneously, and the EB strips were tonically contracted with carbachol. Both tissues relaxed in response to electrical-field stimulation. Phosphorylation of the LC 20 was determined in tissues quick-frozen during relaxation and during stress redevelopment after cessation of field stimulation. Stress and phosphorylation levels were low after 30 s of field stimulation, and a rapid contraction followed field stimulation. Phosphorylation in the LES increased from 0.043 +/- 0.029 to 0.328 +/- 0.043 mol Pi/mol LC 20 within 10 s after stimulation of the inhibitory nerves was terminated, while stress was still rising rapidly. Phosphorylation in the LES then declined to a steady-state value of 0.162 +/- 0.034 mol Pi/mol LC 20 after 10 min. Isotonic shortening velocities at a constant afterload following a quick release showed changes with time that were proportional to the level of phosphorylation. This was also true for values of maximal shortening velocity estimated for zero external load and for the rate of stress redevelopment after a step shortening. Comparable measurements were made in the carbachol-contracted EB. These results indicate that visceral smooth muscles, which normally function tonically (LES) or phasically (EB), exhibit an initial rapid mechanical activation associated with myosin phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Cholinergic nerve-mediated excitation in the guinea pig choledochoduodenal junction activated by distension

1994 ◽  
Vol 267 (5) ◽  
pp. G938-G946 ◽  
Author(s):  
F. Vogalis ◽  
R. R. Bywater ◽  
G. S. Taylor
Keyword(s):  
Smooth Muscle ◽  
Guinea Pig ◽  
Action Potential ◽  
Action Potentials ◽  
Discharge Rate ◽  
Circular Muscle ◽  
Muscle Layer ◽  
Circular Muscle Layer ◽  
Circular Layer ◽  
Longitudinal Layer

The electrical basis of propulsive contractions in the guinea pig choledochoduodenal junction (CDJ), which are triggered by distension, was investigated using intracellular microelectrode recording techniques. The isolated CDJ was placed in a continuously perfused tissue chamber at 37 degrees C. Membrane potential was recorded from smooth muscle cells in either the ampulla or in the upper CDJ (upper junction) regions, which were immobilized by pinning. Distension of the upper junction (20-30 s) by increasing intraductal hydrostatic pressure (mean elevation: 2.0 +/- 0.3 kPa, n = 13) triggered "transient depolarizations" (TDs: < 5 mV in amplitude and 2-5 s in duration) and action potentials in the circular muscle layer of the ampulla. The frequency of TDs in the ampulla was increased from 2.2 +/- 0.2 to 15.9 +/- 2.2 min-1 (n = 13) during distension. Simultaneous impalements of cells in the longitudinal and circular muscle layers in the ampulla revealed that subthreshold TDs in the circular layer were associated with an increased rate of action potential discharge in the longitudinal layer. Atropine (Atr; 1.4 x 10(-6) M) and tetrodotoxin (TTX; 3.1 x 10(-6) M blocked the distension-evoked increase in TD frequency, without affecting the frequency of ongoing TDs. The sulfated octapeptide of cholecystokinin (1-5 x 10(-8) M) increased the amplitude of TDs recorded in the circular muscle layer of the ampulla and increased action potential discharge rate. In separate recordings, radial stretch of the ampulla region increased the rate of discharge of action potentials in the smooth muscle of the upper junction.(ABSTRACT TRUNCATED AT 250 WORDS)

Origin of Motion in the Human Ureter: Mechanics, Energetics and Kinetics of the Myosin Molecular Motors

2012 ◽  
Vol 79 (2) ◽  
pp. 123-129 ◽  
Author(s):  
Romina Vargiu ◽  
Anna Perinu ◽  
Antonello De Lisa ◽  
Frank Tintrup ◽  
Francesco Manca ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Molecular Motors ◽  
Circular Muscle ◽  
Muscle Layer ◽  
Shortening Velocity ◽  
Smooth Muscle Layer ◽  
Circular Smooth Muscle ◽  
Longitudinal Smooth Muscle ◽  
Human Ureter

Background Ureteral peristalsis is the result of coordinated mechanical motor performance of longitudinal and circular smooth muscle layer of the ureter wall. The main aim of this study was to characterize in smooth muscle of proximal segments of human ureter, the mechanical properties at level of muscle tissue and at level of myosin molecular motors. Methods Ureteral samples were collected from 15 patients, who underwent nephrectomy for renal cancer. Smooth muscle strips longitudinally and circularly oriented from proximal segments of human ureter were used for the in vitro experiments. Mechanical indices including the maximum unloaded shortening velocity (Vmax), and the maximum isometric tension (P0) normalized per cross-sectional area, were determined in vitro determined in electrically evoked contractions of longitudinal and circular smooth muscle strips. Myosin cross-bridge (CB) number per mm2 (Ψ) the elementary force per single CB (Ψ) and kinetic parameters were calculated in muscle strips, using Huxley's equations adapted to nonsarcomeric muscles. Results Longitudinal smooth muscle strips exhibited a significantly (p<0.05) faster Vmax (63%) and a higher P0 (40%), if compared to circular strips. Moreover, longitudinal muscle strips showed a significantly higher unitary force (Ψ) per CB. However, no significant differences were observed in CB number, the attachment (f1) and the detachment (g2) rate constants between longitudinal and circular muscle strips. Conclusions The main result obtained in the present work documents that the mechanical, energetic and unitary forces per CB of longitudinal layer of proximal ureter are better compared to the circular one; these preliminary findings suggested, unlike intestinal smooth muscle, a major role of longitudinal smooth muscle layer in the ureter peristalsis.

Proteins of interstitial cells of Cajal and intestinal smooth muscle, colocalized with caveolin-1

2005 ◽  
Vol 288 (3) ◽  
pp. G571-G585 ◽  
Author(s):  
Woo Jung Cho ◽  
E. E. Daniel
Keyword(s):  
Skeletal Muscle ◽  
Smooth Muscle ◽  
Gap Junction ◽  
Interstitial Cells Of Cajal ◽  
Smooth Muscles ◽  
Circular Muscle ◽  
Interstitial Cells ◽  
Caveolin 1 ◽  
Cells Of Cajal ◽  
Junction Proteins

The murine jejunum and lower esophageal sphincter (LES) were examined to determine the locations of various signaling molecules and their colocalization with caveolin-1 and one another. Caveolin-1 was present in punctate sites of the plasma membranes (PM) of all smooth muscles and diffusely in all classes of interstitial cells of Cajal (ICC; identified by c-kit immunoreactivity), ICC-myenteric plexus (MP), ICC-deep muscular plexus (DMP), ICC-serosa (ICC-S), and ICC-intramuscularis (IM). In general, all ICC also contained the L-type Ca2+ (L-Ca2+) channel, the PM Ca2+ pump, and the Na+/Ca2+ exchanger-1 localized with caveolin-1. ICC in various sites also contained Ca2+-sequestering molecules such as calreticulin and calsequestrin. Calreticulin was present also in smooth muscle, frequently in the cytosol, whereas calsequestrin was present in skeletal muscle of the esophagus. Gap junction proteins connexin-43 and -40 were present in circular muscle of jejunum but not in longitudinal muscle or in LES. In some cases, these proteins were associated with ICC-DMP. The large-conductance Ca2+-activated K+ channel was present in smooth muscle and skeletal muscle of esophagus and some ICC but was not colocalized with caveolin-1. These findings suggest that all ICC have several Ca2+-handling and -sequestering molecules, although the functions of only the L-Ca2+ channel are currently known. They also suggest that gap junction proteins are located at sites where ultrastructural gap junctions are know to exist in circular muscle of intestine but not in other smooth muscles. These findings also point to the need to evaluate the function of Ca2+ sequestration in ICC.

Variability in the muscle composition of rat esophagus and neural pathway of lower esophageal sphincter relaxation

2011 ◽  
Vol 301 (6) ◽  
pp. G1014-G1019 ◽  
Author(s):  
Yanfen Jiang ◽  
Valmik Bhargava ◽  
Harshal A. Lal ◽  
Ravinder K. Mittal
Keyword(s):  
Skeletal Muscle ◽  
Smooth Muscle ◽  
Vagus Nerve ◽  
Longitudinal Muscle ◽  
Muscle Layer ◽  
Axial Stretch ◽  
Longitudinal Muscle Layer ◽  
Esophageal Sphincter ◽  
Group 2 ◽  
Group 1

Several studies from our laboratory show that axial stretch of the lower esophageal sphincter (LES) in an oral direction causes neurally mediated LES relaxation. Under physiological conditions, axial stretch of the LES is caused by longitudinal muscle contraction (LMC) of the esophagus. Because longitudinal muscle is composed of skeletal muscle in mice, vagal-induced LMC and LES relaxation are both blocked by pancuronium. We conducted studies in rats (thought to have skeletal muscle esophagus) to determine if vagus nerve-mediated LES relaxation is also blocked by pancuronium. LMC-mediated axial stretch on the LES was monitored using piezoelectric crystals. LES and esophageal pressures were monitored with a 2.5-Fr solid-state pressure transducer catheter. Following bilateral cervical vagotomy, the vagus nerve was stimulated electrically. LES, along with the esophagus, was harvested after in vivo experiments and immunostained for smooth muscle (smooth muscle α-actin) and skeletal muscle (fast myosin heavy chain). Vagus nerve-stimulated LES relaxation and esophageal LMC were reduced in a dose-dependent fashion and completely abolished by pancuronium (96 μg/kg) in six rats ( group 1). On the other hand, in seven rats, LES relaxation and LMC were only blocked completely by a combination of pancuronium ( group 2) and hexamethonium. Immunostaining revealed that the longitudinal muscle layer was composed of predominantly skeletal muscle in the group 1 rats. On the other hand, the longitudinal muscle layer of group 2 rats contained a significant amount of smooth muscle ( P < 0.05). Our study shows tight coupling between axial stretch on the LES and relaxation of the LES, which suggests a cause and effect relationship between the two. We propose that the vagus nerve fibers that cause LMC induce LES relaxation through the stretch-sensitive activation of inhibitory motor neurons.

scholarly journals Rhythmic Electrical Activity in Stomach and Intestine of Toad

1980 ◽  
Vol 86 (1) ◽  
pp. 237-248
Author(s):  
ALLEN MANGEL ◽  
C. LADD PROSSER
Keyword(s):  
Smooth Muscle ◽  
Longitudinal Muscle ◽  
Circular Muscle ◽  
Sodium Concentration ◽  
Muscle Layer ◽  
Free Solution ◽  
Longitudinal Muscle Layer ◽  
Longitudinal Smooth Muscle ◽  
Mammalian Intestine ◽  
Periodic Changes

The intact stomach of the toad initiates rhythmic slow-spikes of 5–15 s duration and frequency of 3-5 min−1. The spontaneous electrical waves originate in the longitudinal muscle layer; isolated circular muscle is quiescent. Aboral conduction velocity is 0.12–0.9 mm s−1. Reduction of external sodium concentration from 89.5 to 15 mM produced no effect on slow spikes, although further reduction to 1.5 mM increased frequency and decreased amplitude. Slow-spikes were unaffected by ouabain or by incubation in potassium-free solution. When calcium in the medium was reduced, slow-spike amplitude and frequency decreased. Slow-spikes exhibited a change in amplitude of 16 mV per decade change in CaO2+; slow-spikes were eliminated at 10−8 M CaO2+ and by blockers of calcium conductance channels. Intact intestine of toad demonstrated slow-waves which resembled those of mammalian intestine. These were sensitive to changes in external sodium and were eliminated by 1 × 10−4M ouabain. It is suggested that rhythmic slow-spikes of longitudinal smooth muscle of amphibian stomach may result from periodic changes in Ca conductance whereas endogenous electrical waves of intestine may result from rhythmic extrusion of sodium.

Membrane potential gradient is carbon monoxide-dependent in mouse and human small intestine

2007 ◽  
Vol 293 (2) ◽  
pp. G438-G445 ◽  
Author(s):  
Lei Sha ◽  
Gianrico Farrugia ◽  
W. Scott Harmsen ◽  
Joseph H. Szurszewski
Keyword(s):  
Carbon Monoxide ◽  
Smooth Muscle ◽  
Small Intestine ◽  
Smooth Muscle Cells ◽  
Circular Muscle ◽  
Muscle Cells ◽  
Muscle Layer ◽  
Wild Type ◽  
Circular Smooth Muscle ◽  
Human Small Intestine

The aims of this study were to quantify the change in resting membrane potential (RMP) across the thickness of the circular muscle layer in the mouse and human small intestine and to determine whether the gradient in RMP is dependent on the endogenous production of carbon monoxide (CO). Conventional sharp glass microelectrodes were used to record the RMPs of circular smooth muscle cells at different depths in the human small intestine and in wild-type, HO2-KO, and W/WV mutant mouse small intestine. In the wild-type mouse and human intestine, the RMP of circular smooth muscle cells near the myenteric plexus was −65.3 ± 2 mV and −58.4 ± 2 mV, respectively, and −60.1 ± 2 mV and −49.1 ± 1 mV, respectively, in circular smooth muscle cells at the submucosal border. Oxyhemoglobin (20 μM), a trapping agent for CO, and chromium mesoporphyrin IX, an inhibitor of heme oxygenase, abolished the transwall gradient. The RMP gradients in mouse and human small intestine were not altered by NG-nitro-l-arginine (200 μM). No transwall RMP gradient was found in HO2-KO mice and W/WV mutant mice. TTX (1 μM) and 1H-[1,2,4-]oxadiazolo[4,3-a]quinoxalin-1-one (10 μM) had no effect on the RMP gradient. These data suggest that the gradient in RMP across the thickness of the circular muscle layer of mouse and human small intestine is CO dependent.

Neuromuscular structures specific to the submucosal border of the human colonic circular muscle layer

1990 ◽  
Vol 68 (11) ◽  
pp. 1437-1446 ◽  
Author(s):  
M. S. Faussone-Pellegrini ◽  
C. Cortesini ◽  
D. Pantalone
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Interstitial Cells Of Cajal ◽  
Circular Muscle ◽  
Muscle Cells ◽  
Muscle Layer ◽  
Interstitial Cells ◽  
Circular Muscle Layer ◽  
The Right ◽  
Cells Of Cajal

The circular muscle layer of the human caecum and ascending colon is clearly subdivided into two portions: an outer one which includes the bulk of the circular muscle layer, and an inner one made up of only six to eight rows of cells. In the right transverse colon no demarcation can be observed, but a difference exists between the innermost and the outermost cells, since those of the two innermost rows possess some peculiarities with regard to the sarcoplasmic reticulum, glycogen particles, caveolae, and intercellular junctions. In the left part of the colon, the circular muscle layer is also divided into two portions. In fact, the innermost smooth muscle cells still possess peculiar morphologies, progressively increase in number, and become separate from each other making up a superficial muscle network. A fibrous lamella, along and inside which a ganglionated nerve plexus runs, is strictly apposed to the submucosal border of the circular muscle layer of the entire colonic length. A second nerve plexus runs between the two portions of the circular muscle layer. Both these plexuses are accompanied by interstitial cells of Cajal in the right colon only. The peculiar organization of the entire submucosal border of the human colonic circular muscle layer distinguishes it from other parts of the gut and probably represents a structural basis for control of human colonic motility. The presence of putative pacemaker cells (interstitial cells and peculiar smooth muscle cells) indicates that the inner border of human colonic circular muscle layer possesses pacemaking activities. Moreover, the interstitial cell – smooth muscle cell ratio differs depending on the colonic level; two main regions can be identified: the right and the left colon. Consequently, we might expect regional variation in pacemaking.Key words: smooth muscle cells, interstitial cells of Cajal, human colon, ultrastructure.

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