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.

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 Limited Ability of Posaconazole To Cure both Acute and Chronic Trypanosoma cruzi Infections Revealed by Highly SensitiveIn VivoImaging

2015 ◽  
Vol 59 (8) ◽  
pp. 4653-4661 ◽  
Author(s):  
Amanda Fortes Francisco ◽  
Michael D. Lewis ◽  
Shiromani Jayawardhana ◽  
Martin C. Taylor ◽  
Eric Chatelain ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
Imaging System ◽  
Ex Vivo ◽  
Parasite Burden ◽  
Significant Activity ◽  
Content Type ◽  
Chronic Stage

ABSTRACTThe antifungal drug posaconazole has shown significant activity againstTrypanosoma cruziin vitroand in experimental murine models. Despite this, in a recent clinical trial it displayed limited curative potential. Drug testing is problematic in experimental Chagas disease because of difficulties in demonstrating sterile cure, particularly during the chronic stage of infection when parasite burden is extremely low and tissue distribution is ill defined. To better assess posaconazole efficacy against acute and chronic Chagas disease, we have exploited a highly sensitive bioluminescence imaging system which generates data with greater accuracy than other methods, including PCR-based approaches. Mice inoculated with bioluminescentT. cruziwere assessed byin vivoandex vivoimaging, with cyclophosphamide-induced immunosuppression used to enhance the detection of relapse. Posaconazole was found to be significantly inferior to benznidazole as a treatment for both acute and chronicT. cruziinfections. Whereas 20 days treatment with benznidazole was 100% successful in achieving sterile cure, posaconazole failed in almost all cases. Treatment of chronic infections with posaconazole did however significantly reduce infection-induced splenomegaly, even in the absence of parasitological cure. The imaging-based screening system also revealed that adipose tissue is a major site of recrudescence in mice treated with posaconazole in the acute, but not the chronic stage of infection. Thisin vivoscreening model for Chagas disease is predictive, reproducible and adaptable to diverse treatment schedules. It should provide greater assurance that drugs are not advanced prematurely into clinical trial.

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 Type I Interferons Increase Host Susceptibility to Trypanosoma cruzi Infection

2011 ◽  
Vol 79 (5) ◽  
pp. 2112-2119 ◽  
Author(s):  
Anne-Danielle C. Chessler ◽  
Kacey L. Caradonna ◽  
Akram Da'dara ◽  
Barbara A. Burleigh
Keyword(s):  
Trypanosoma Cruzi ◽  
Parasite Burden ◽  
Host Susceptibility ◽  
Type I Interferons ◽  
Type I ◽  
Type I Ifn ◽  
Content Type ◽  
Type I Ifns ◽  
Ifn Γ ◽  
The Impact

ABSTRACTTrypanosoma cruzi, the protozoan parasite that causes human Chagas' disease, induces a type I interferon (IFN) (IFN-α/β) response during acute experimental infection in mice and in isolated primary cell types. To examine the potential impact of the type I IFN response in shaping outcomes in experimentalT. cruziinfection, groups of wild-type (WT) and type I IFN receptor-deficient (IFNAR−/−) 129sv/ev mice were infected with two differentT. cruzistrains under lethal and sublethal conditions and several parameters were measured during the acute stage of infection. The results demonstrate that type I IFNs are not required for early host protection againstT. cruzi. In contrast, under conditions of lethalT. cruzichallenge, WT mice succumbed to infection whereas IFNAR−/−mice were ultimately able to control parasite growth and survive.T. cruziclearance in and survival of IFNAR−/−mice were accompanied by higher levels of IFN-γ production by isolated splenocytes in response to parasite antigen. The suppression of IFN-γ in splenocytes from WT mice was independent of IL-10 levels. While the impact of type I IFNs on the production of IFN-γ and other cytokines/chemokines remains to be fully determined in the context ofT. cruziinfection, our data suggest that, under conditions of high parasite burden, type I IFNs negatively impact IFN-γ production, initiating a detrimental cycle that contributes to the ultimate failure to control infection. These findings are consistent with a growing theme in the microbial pathogenesis field in which type I IFNs can be detrimental to the host in a variety of nonviral pathogen infection models.

scholarly journals Trypanosoma cruzi Causes Paralyzing Systemic Necrotizing Vasculitis Driven by Pathogen-Specific Type I Immunity in Mice

2016 ◽  
Vol 84 (4) ◽  
pp. 1123-1136 ◽  
Author(s):  
Ester Roffê ◽  
Ana Paula M. P. Marino ◽  
Joseph Weaver ◽  
Wuzhou Wan ◽  
Fernanda F. de Araújo ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Trypanosoma Cruzi ◽  
Acute Infection ◽  
Chronic Inflammatory Disease ◽  
Interleukin 17 ◽  
Type I ◽  
Necrotizing Vasculitis ◽  
Necrosis Factor Alpha ◽  
Content Type ◽  
Inflammatory Monocytes

Infectious agents are often considered potential triggers of chronic inflammatory disease, including autoimmunity; however, direct evidence is usually lacking. Here we show that following control of acute infection of mice with the myotropic Colombiana strain ofTrypanosoma cruzi, parasites persisted in tissue at low levels associated with development of systemic necrotizing vasculitis. Lesions occurred in many but not all organs and tissues, with skeletal muscle arteries being the most severely affected, and were associated with myositis, atrophy, paresis/paralysis, and death. Histopathology showed fibrinoid vascular necrosis, rare amastigote nests within skeletal muscle myocytes, and massive leukocyte infiltrates composed mainly of inflammatory monocytes, F4/80+macrophages, andT. cruzitetramer-specific CD8+T lymphocytes capable of producing gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) but not interleukin-17 (IL-17).T. cruzi-specific IgG was detected in sera from infected mice, but antibody deposits and neutrophilic inflammation were not features of the lesions. Thus,T. cruziinfection of mice may be a specific infectious trigger of paralyzing systemic necrotizing vasculitis most severely affecting skeletal muscle, driven by pathogen-specific type I immune responses.

Sign in / Sign up

Export Citation Format

Share Document