Effect of mast cell stabilization on angiogenesis in primary and secondary experimental Trichinella spiralis infection.

BACKGROUND: Mast cells are known to affect the primary and secondary immune responses against parasites, and this effect is partially mediated through the release of pro-angiogenic mediators. The aim of this study was to explore the effect of the mast cell stabilizer (MCS), ketotifen, with and without albendazole, an anti-parasitic prescription medicine, on the inflammatory response against Trichinella spiralis, with the overall aim to investigate its effect on angiogenesis accompanying nurse cell formation. METHODS: The effect of ketotifen and albendazole was explored in eight groups of female BALB/c mice. Four groups were sensitized with a small dose of T. spiralis larvae. The drug regimen was then applied to both sensitized (challenged) and non-sensitized mice. The parasite load was assessed by histopathological examination of the small intestine and muscle tissue, and angiogenesis was assessed by immunohistochemistry to determine the expression of vascular endothelial growth factor (VEGF). RESULTS: Sensitized mice showed a significantly lower parasite load and a more pronounced inflammatory response than mice receiving a single infective dose of T. spiralis larvae. All treated groups showed a significant reduction in parasite count compared to the control groups (groups IAa and IBa), reaching approximately an 98.8% reduction in adult parasite count in the sensitized group treated with albendazole (groups IIAb and IIBb). MCS significantly decreased the parasite count during both the intestinal or muscular phases, reduced tissue inflammation, and decreased local VEGF expression, both in the non-sensitized and sensitized groups. CONCLUSION: Sensitization with a low dose of T. spiralis larvae was found to confer a partial protective immunity against re-infection and to positively affect the study outcomes, thus underlining the importance of vaccination, but after extensive studies. The anti-angiogenic effect of MCS protects against larval encystation during the muscle phase. The anti-angiogenic potential of albendazole suggests that the action of this anti-helminthic during trichinellosis is not confined to structural damage to the parasite cuticle but includes an effect on host immunopathological response.

Myocarditis and Raw Meat Consumption: Strange Bedfellows!

Trichinellosis is a parasitic infection that is associated with the consumption of raw meat. The specific genotype Trichinella nativa has been found in raw bear meat. The most common genotype that has been linked with myocarditis is T spiralis. We present a case of T nativa myocarditis secondary to consumption of raw bear meat. The clinical manifestations as well as therapy of this specific genotype is outlined.

The effect of Trichinella spiralis on muscular activity of experimentally infected mice.

Nematodes of genus Trichinella are wide-spread zoonotic parasites, able to infect a large variety of vertebrates. Animal hosts are usually regarded as asymptomatic carriers. However, there is little data regarding the functional consequences that T. spiralis infection renders on muscle cells. The aim of the present study was to assess the effect of T. spiralis on the effort capacity of experimentally infected mice. Overall, 60 mice, divided into three groups were used: M (uninfected), L200 and L1000, infected with 200 or 1000 larvae/mouse respectively. The mice were periodically weighed and their effort capacity was evaluated (days 0, 7, 15, 35 and 60). From each group, two randomly selected mice were euthanized after evaluation carcasses were artificially digested in order to establish the number of larvae per gram (LPG). On day 0, there were no significant differences among groups. Starting with day 7, the effort capacity of infected groups decreased, with significant differences between group M and the infected groups. From day 15, the differences between the infected groups also became significant. The LPG gradually increased and the differences between groups were always significant. A strong correlation between the LPG and decreased effort capacity was noted. The present study demonstrates the reduction of muscular capacity in mice experimentally infected with Trichinella spiralis, in correlation with the infective dose, providing new insights in this parasite's transmission strategy.

Label-free quantitative proteomic analysis of molting-related proteins of Trichinella spiralis intestinal infective larvae.

Molting is a key step for body-size expansion and environmental adaptation of parasitic nematodes, and it is extremely important for Trichinella spiralis growth and development, but the molting mechanism is not fully understood. In this work, label-free LC-MS/MS was used to determine the proteome differences between T. spiralis muscle larvae (ML) at the encapsulated stage and intestinal infective larvae (IIL) at the molting stage. The results showed that a total of 2885 T. spiralis proteins were identified, 323 of which were differentially expressed. These proteins were involved in cuticle structural elements, regulation of cuticle synthesis, remodeling and degradation, and hormonal regulation of molting. These differential proteins were also involved in diverse intracellular pathways, such as fatty acid biosynthesis, arachidonic acid metabolism, and mucin type O-glycan biosynthesis. qPCR results showed that five T. spiralis genes (cuticle collagen 14, putative DOMON domain-containing protein, glutamine synthetase, cathepsin F and NADP-dependent isocitrate dehydrogenase) had significantly higher transcriptional levels in 10 h IIL than ML (P < 0.05), which were similar to their protein expression levels, suggesting that they might be T. spiralis molting-related genes. Identification and characterization of T. spiralis molting-related proteins will be helpful for developing vaccines and new drugs against the early enteral stage of T. spiralis.

Identification of a host collagen inducing factor from the excretory secretory proteins of Trichinella spiralis.

BACKGROUND: In a previous study, we found that Trichinella spiralis muscle larva excretory and secretory proteins (ES-P) most likely activate collagen synthesis via TGF-ß/Smad signaling, and this event could influence collagen capsule formation. METHODOLOGY/PRINCIPAL FINDINGS: In order to identify the specific collagen inducing factor, ES-P was fractionated by a Superdex 200 10/300 GL column. We obtained three large fractions, F1, F2, and F3, but only F3 had collagen gene inducing ability. After immunoscreening, 10 collagen inducing factor candidates were identified. Among them, TS 15-1 and TS 15-2 were identical to the putative trypsin of T. spiralis. The deduced TS 15-1 (M.W. = 72 kDa) had two conserved catalytic motifs, an N-terminal Tryp_SPc domain (TS 15-1n) and a C-terminal Tryp_SPc domain (TS 15-1c). To determine their collagen inducing ability, recombinant proteins (rTS 15-1n and rTS 15-1c) were produced using the pET-28a expression system. TS 15-1 is highly expressed during the muscle larval stage and has strong antigenicity. We determined that rTS 15-1c could elevate collagen I via activation of the TGF-ß1 signaling pathway in vitro and in vivo. CONCLUSION/SIGNIFICANCE: In conclusion, we identified a host collagen inducing factor from T. spiralis ES-P using immunoscreening and demonstrated its molecular characteristics and functions.

Effect of Muscle Strength by Trichinella spiralis Infection during Chronic Phase.

Introduction:Trichinella spiralis establishes a chronic infection in skeletal muscle by developing nurse cells within muscle fibers. During symbiosis in host, changes in the muscle fibers and inflammation may affect muscle function. Methods: We investigated muscle strength and inflammation in T. spiralis-infected mice during 1 to 48 weeks after infection. Results: Muscle strength decreased compared to that in uninfected control mice during the late infection stage. Additionally, inflammatory related cytokines increased significantly during early stage of infection and then rapidly decreased. In pathological study, nuclear infiltration maintained from the early infection stage to chronic infection stage. Moreover, vacuoles and eosinophil infiltration were observed in infected muscle in chronic stage. Conclusion: These results suggest that infection by T. spiralis significantly affects muscle function was continuously being weakness because vacuoles formation and maintained nucleus and eosinophil infiltration during chronic phase of T. spiralis infection.

Oral contraceptive pills: Risky or protective in case of Trichinella spiralis infection?

The aim of this study was to investigate how Trichinella spiralis infection can be affected by contraceptive pills in vivo. Methods included six groups of female Wistar rats; healthy, Trichinella infected, receiving combined contraceptive pills (COCPs), receiving progestin only pills (POPs), infected receiving COCPs and infected receiving POPs. Parasite burden was measured; adult worm counts, gravidity, larvae and reproductive capacity index). Histopathological examination, immunohistochemical detection of C-kit+ mast cells and Foxp3+ T-reg. cells in intestinal sections, eosinophils muscle infiltration and CPK level were performed. Rats infected and receiving COCPs showed a significant increase in parasitic burden, and infected receiving POPs showed a significant reduction compared to infected only, with a significant increase in nongravid females (Mean total worms=964.40±55.9, 742±52.63, 686±31.68, larvae/g=5030±198.75, 2490±143.18 and 4126±152,91, respectively). Intestinal sections from infected receiving COCPs showed intact mucosa (though the high inflammatory cells infiltrate), and significant increase in C-kit+ mast cells number and intensity (30.20±4.15 and 60.40±8.29), and Foxp3+ T-reg. cells (10±1.58). Infected receiving POPs showed a significantly less CPK (5886±574.40) and eosinophilic muscle infiltration (58±13.51). Oestrogen-containing pills established a favourable intestinal environment for Trichinella by enhancing Foxp+T-reg. cells and stabilizing C-kit+mast cells, while POPs gave a potential protection with less gravidity, larval burden and eosinophilic infiltrate.

The induction of the collagen capsule synthesis by Trichinella spiralis is closely related to protease-activated receptor 2.

The muscle-stage larvae of the parasite Trichinella spiralis have the ability to survive within host muscle tissue by virtue of the formation a nurse cell-parasite complex, which is surrounded by collagen. The formation of the complex is initiated by excretory-secretory (ES) proteins produced by the parasite. To determine the mechanisms underlying collagen capsule formation, we investigated the expression levels of several types of collagen genes and TGF-ßI signaling-related genes (Smad2 and Smad3) in muscle cells. Synthesis of type I, IV, and VI collagen, which are major constituents of the collagen capsule, significantly increased during T. spiralis infection. In addition, we found that expression of the protease-activated receptor 2 (PAR2) gene was significantly increased during this period. Expression levels of the collagen genes and TGF-ßI, Smad2, and Smad3 were induced by ES proteins and a PAR2 agonist, whereas their enhanced expression levels were reduced by a PAR2 antagonist and serine protease inhibitors. To evaluate the involvement of PAR2 during T. spiralis infection in vivo, we infected wild-type and PAR2 knockout (KO) mice with T. spiralis. Expression levels of type I, IV, and VI collagen genes and TGF-ßI signaling-related genes (Smad2 and Smad3) were also decreased in the PAR2 KO mice. Phosphorylation of Smad2/3, which was increased by T. spiralis infection, was significantly diminished in the PAR2 KO mice. In conclusion, ES proteins containing serine protease most likely activate collagen synthesis via PAR2 and TGF-ßI signaling, and this event could influence collagen capsule formation.

Changes in Epithelial Barrier Function in Response to Parasitic Infection: Implications for IBD Pathogenesis.

BACKGROUND AND AIMS: Mast cells [MCs] are implicated in epithelial barrier alterations that characterize inflammatory and functional bowel disorders. In this study, we describe mast cell proteinases [chymases and tryptases] and tight junction [TJ] proteins kinetics in a rat model of postinfectious gut dysfunction. METHODS: Jejunal tissues of control and -infected rats were used. Inflammation-related changes in MCs and the expression of TJ-related proteins were evaluated by immunostaining and reverse transcription-quantitative polymerase chain reaction. Epithelial barrier function was assessed in vitro (Ussing chambers) and in vivo. RESULTS: After infection, intestinal inflammation was associated with a generalized overexpression of MC chymases, peaking between Days 6 and 14. Thereafter, a mucosal MC hyperplasia and a late increase in connective tissue MC counts were observed. From Day 2 post-infection, TJ proteins occludin and claudin-3 expression was down-regulated whereas the pore-forming protein claudin-2 was overexpressed. The expression of proglucagon, precursor of the barrier-enhancing factor glucagon-like peptide-2, was reduced. These changes were associated with an increase in epithelial permeability, both in vitro and in vivo. CONCLUSIONS: Proteinases expression and location of mucosal and connective tissue MCs indicate a time-related pattern in the maturation of intestinal MCs following infection. Altered expression of TJ-related proteins is consistent with a loss of epithelial tightness, and provides a molecular mechanism for the enhanced epithelial permeability observed in inflammatory conditions of the gut.

[A MATHEMATICAL MODEL FOR ESTIMATING THE SEVERITY OF EXPERIMENTAL TRICHINOSIS].

The authors propose a mathematical model that makes it possible to estimate the severity of experimental trichinosis from the peripheral blood values of a laboratory animal (white blood cells, neutrophils, eosinophils, and lymphocyates).

[On the possibility of vertical transmission of Trichinella and the impact of its invasion].

Whether experimentally infected female rats can transmit Trichinella in utero and its invasion may affect the immunity of their offspring has been investigated. There is evidence that maternal antibodies have a significant impact on the humoral immunity of the offspring and as a result on the number of Trichinella larvae accustomed in the muscles. Immune interlayer formation in the populations of carnivorous mammals in the endemic areas occurs due to reinvasions and passive transmission of maternal antibodies to the offspring. High-tension immunity in the population and animals contributes to a reduction in the onset of invasion and to a predominance of the subclinical course of the disease and asymptomatic carriage in a specific focus. Passive immunity transmitted from the Trichinella-infected mother to its offspring is of importance in the development of epidemic and epizootic processes in the natural and synanthropic foci of trichinosis.

Immune response to a Trichinella spiralis infection in house mice from lines selectively bred for high voluntary wheel running.

Four lines of mice bred for high voluntary wheel running (HR lines) have high baseline circulating corticosterone levels and increased daily energy expenditure as compared with four non-selected control (C) lines. High corticosterone may suppress immune function and competing energy demands may limit ability to mount an immune response. We hypothesized that HR mice have a reduced immune response and therefore a decreased ability to fight an infection by Trichinella spiralis, an ecologically relevant nematode common in mammals. Infections have an acute, intestinal phase while the nematode is migrating, reproducing and traveling throughout the bloodstream, followed by a chronic phase with larvae encysted in muscles. Adult males (generation 55 of the selection experiment) were sham-infected or infected by oral gavage with ~300 J1 T. spiralis larvae. During the chronic phase of infection, mice were given wheel access for 6 days, followed by 2 days of maximum aerobic performance trials. Two weeks post-infection, infected HR had significantly lower circulating immunoglobulin E levels compared with infected C mice. However, we found no statistical difference between infected HR and C mice in numbers of encysted larvae within the diaphragm. As expected, both voluntary running and maximum aerobic performance were significantly higher in HR mice and lower in infected mice, with no line type-by-infection interactions. Results complement those of previous studies suggesting decreased locomotor abilities during the chronic phase of T. spiralis infection. However, despite reduced antibody production, breeding for high voluntary wheel exercise does not appear to have a substantial negative impact on general humoral function.

PAR-2-mediated control of barrier function and motility differs between early and late phases of postinfectious gut dysfunction in the rat.

Proteinase-activated receptor-2 (PAR-2) and mast cell (MC) mediators contribute to inflammatory and functional gastrointestinal disorders. We aimed to characterize jejunal PAR-2-mediated responses and the potential MC involvement in the early and late phases of a rat model of postinfectious gut dysfunction. Jejunal tissues of control and Trichinella spiralis-infected (14 and 30 days postinfection) rats, treated or not with the MC stabilizer, ketotifen, were used. Histopathology and immunostaining were used to characterize inflammation, PAR-2 expression, and mucosal and connective tissue MCs. Epithelial barrier function (hydroelectrolytic transport and permeability) and motility were assessed in vitro in basal conditions and after PAR-2 activation. Intestinal inflammation on day 14 postinfection (early phase) was significantly resolved by day 30 (late phase) although MC counts and epithelial permeability remained increased. PAR-2-mediated ion transport (Ussing chambers, in vitro) and epithelial surface PAR-2 expression were reduced in the early phase, with a trend toward normalization during the late phase. In control conditions, PAR-2 activation (organ bath) induced biphasic motor responses (relaxation followed by excitation). At 14 days postinfection, spontaneous contractility and PAR-2-mediated relaxations were enhanced; motor responses were normalized on day 30. Postinfectious changes in PAR-2 functions were not affected by ketotifen treatment. We concluded that, in the rat model of Trichinella spiralis infection, alterations of intestinal PAR-2 function and expression depend on the inflammatory phase considered. A lack of a ketotifen effect suggests no interplay between MCs and PAR-2-mediated motility and ion transport alterations. These observations question the role of MC mediators in PAR-2-modulating postinfectious gut dysfunction.

Persistent epithelial barrier alterations in a rat model of postinfectious gut dysfunction.

BACKGROUND: Mucosal mast cells (MMCs), epithelial barrier function (EBF) and the enteric nervous system (ENS) are interactive factors in the pathophysiology of functional gastrointestinal disorders. We characterized postinfectious EBF alterations in the Trichinella spiralis infection model of MMC-dependent intestinal dysfunction in rats. METHODS: Sprague-Dawley rats were infected with T. spiralis. 30 ± 2 days postinfection, jejunal EBF (electrophysiological parameters, fluorescein isothiocyanate-dextran fluxes and responses to secretagogues and MMC degranulators) was evaluated (Ussing chamber). In some experiments, participation of secretomotor neurons was examined by tetrodotoxin (TTX) pretreatment. Jejunal histology and MMC count and activity were also assessed. KEY RESULTS: 30 ± 2 days postinfection, when only a low grade inflammation was observed, increased MMC number and activity were associated with altered EBF. EBF alterations were characterized by increased mucosal permeability and ion secretion. In T. spiralis-infected animals, secretory responses to serotonin (5-HT) and immunoglobulin E (IgE)-dependent activation of MMCs were reduced. In contrast, responses to substance P (SP) and capsaicin were similar in infected and noninfected animals. Neuronal blockade with TTX altered secretory responses to SP and capsaicin only in infected rats. CONCLUSIONS & INFERENCES: Trichinella spiralis infection in rats, at late stages, results in persistent postinfectious intestinal barrier dysfunctions and mucosal mastocytosis, with other signs suggestive of a low grade inflammation. The altered permeability and the TTX-independent hyporesponsiveness to 5-HT and IgE indicate epithelial alterations. Changes in responses to SP and capsaicin after neuronal blockade suggest an ENS remodeling during this phase. Similar long-lasting neuro-epithelial alterations might contribute to the pathophysiology of functional and postinfectious gastrointestinal disorders.

Pathogenesis of restricted movements in trichinellosis: an experimental study.

Trichinellosis is a zoonosis acquired by the ingestion of insufficiently cooked pork meat containing the encapsulated larvae of Trichinella spiralis. Trichinellosis is presented with myalgia which affects various muscle groups; its intensity is usually related to the severity of the disease and may cause restriction of joint movement. However, joint pain in the course of trichinellosis could not be explained entirely by myositis. This study investigated the other possible causes of restricted movements of joints in animal model. We found that the histopathological changes in the joints of T. spiralis infected rats were in the form of inflammatory cellular infiltrates and ulceration in the synovial membrane with degeneration and ulceration of the articular cartilage. Immunohistochemical examination of the joints revealed the presence of T. spiralis local antigen or immune complex deposited in the synovial membrane. Leukocytosis and eosinophilia were observed throughout the experimental period but eosinophil level declined slowly but still elevated. In conclusion, the restricted movements during the course of trichinellosis seem to be not only due to direct invasion of muscles by the encapsulated T. spiralis larvae but also due to immune complex deposition in the joints.

Reviews on trichinellosis (IV): hepatic involvement.

Trichinella sp. larvae are released from the intestine and usually migrate through the liver into the general circulation. Nevertheless, hepatic involvement does not represent a leading feature of human trichinellosis. This narrative systematic review focuses exclusively on hepatic complications of trichinellosis and includes a brief overview and analysis of selected cases reported in the literature. Our major goals were to gather important data to understand the substrate of trichinous liver injury and to provide helpful clinical hints for practitioners. To our knowledge, this is the first international review dedicated to this topic. Thirty two cases were detailed enough to allow pooled analysis. The mean age of the analyzed group was 30.5±17 years old with most cases aged 19 to 29 years (29%). Hepatomegaly was noticed in 34.4% of cases, hypoalbuminemia in 40.6%, and hypoproteinemia in 34.4%. Our systematic analyses demonstrated that hepatic complications were mainly evidenced in fatal cases of trichinellosis (65.6%). The most frequent necroptic findings were parenchymatous and/or fatty degeneration of the liver (59.4%). Although less discussed in the literature than other complications, hepatic involvement should be considered more carefully because of its significant consequences on protein metabolism and liver function. Difficult to be revealed intra vitam, hepatic involvement may represent an additional concern to the classical clinical picture of trichinellosis and other life-threatening complications, especially in severe cases of infection. Consequently, physicians should be aware of any possible liver involvement to ensure adequate management of their patients.

Reviews on trichinellosis (I): renal involvement.

Besides cardiac and neurological complications of trichinellosis, renal involvement is the most important as regards the cases published and discussed in literature and is generally characteristic of the severe forms of the disease. This article focuses exclusively on the renal disturbances that may occur in this parasitic illness and includes a synopsis of published cases. Our primary goal was to increase the awareness of infectious diseases specialists, nephrologists, and general practitioners about these complications with possible fatal outcome. To our knowledge, this is the first international review dedicated to this topic. Cases for which enough details were available have been analyzed, and the results indicated that the mean age of the affected patients was 31.6 years, with a slightly male predominance (56.2%). The fatality rate was 26.1%. Proteinuria was detected in 84.8% of cases, hematuria in 30.4%, and casts were observed in urine specimens from 23.9% of patients. Renal failure was evidenced in 8.7% of patients, whereas renal lesions were found by biopsy or necropsy in 43.5% of cases. Of the routine laboratory parameters that are relevant for trichinellosis, mean eosinophil count was 32.2% and mean leukocyte count was 17,312 cells/µL. Finally, we emphasize on the necessity of establishing an early and correct diagnosis of trichinellosis to avoid later and severe complications. Additionally, implementation of public health and food safety prophylactic measures against the disease must represent an immediate priority for the affected regions.