Wnt signaling in regulation of biological functions of the nurse cell harboring Trichinella spp.

BACKGROUND: The nurse cell (NC) constitutes in mammalian skeletal muscles a confined intracellular niche to support the metabolic needs of muscle larvae of Trichinella spp. encapsulating species. The main biological functions of NC were identified as hypermitogenic growth arrest and pro-inflammatory phenotype, both inferred to depend on AP-1 (activator protein 1) transcription factor. Since those functions, as well as AP-1 activity, are known to be regulated among other pathways, also by Wnt (Wingless-Type of Mouse Mammary Tumor Virus Integration Site) signaling, transcription profiling of molecules participating in Wnt signaling cascades in NC, was performed. METHODS: Wnt signaling-involved gene expression level was measured by quantitative RT-PCR approach with the use of Qiagen RT(2) Profiler PCR Arrays and complemented by that obtained by searching microarray data sets characterizing NC transcriptome. RESULTS: The genes involved in inhibition of canonical Wnt/ß-catenin signaling cascade as well as leading to ß-catenin degradation were found expressed in NC at high level, indicating inhibition of this cascade activity. High expression in NC of genes transmitting the signal of Wnt non-canonical signaling cascades leading to activation of AP-1 transcription factor, points to predominant role of non-canonical Wnt signaling in a long term maintenance of NC biological functions. CONCLUSIONS: Canonical Wnt/ß-catenin signaling cascade is postulated to play a role at the early stages of NC formation when muscle regeneration process is triggered. Following mis-differentiation of infected myofiber and setting of NC functional specificity, are inferred to be controlled among other pathways, by Wnt non-canonical signaling cascades.

The vasculature of nurse cells infected with non-encapsulated Trichinella species.

The vasculature surrounding the nurse cells of encapsulated Trichinella spiralis has been described previously. It has been postulated the function of these vessels is to support the growth of the parasite. We describe here for the first time the vasculature surrounding the nurse cells of non-encapsulated T. pseudospiralis and T. papuae. Similar to the vasculature of uninfected muscle cells, the vessels surrounding non-encapsulated Trichinella nurse cells are dense and branched longitudinally along the long axis of the muscle cells; they also appear to be similar in diameter. The netting pattern of enlarged vessels found around T. spiralis (encapsulated) nurse cells is not present in non-encapsulated Trichinella infections. The vessels surrounding non-encapsulated Trichinella nurse cells seem to exist prior to parasite invasion of the muscle cell.

New method to disaggregate and analyze single isolated helminthes cells using flow cytometry: proof of concept.

In parasitology, particularly in helminthes studies, several methods have been used to look for the expression of specific molecules, such as RT-PCR, western blot, 2D-electrophoresis, and microscopy, among others. However, these methods require homogenization of the whole helminth parasite, preventing evaluation of individual cells or specific cell types in a given parasite tissue or organ. Also, the extremely high interaction between helminthes and host cells (particularly immune cells) is an important point to be considered. It is really hard to obtain fresh parasites without host cell contamination. Then, it becomes crucial to determine that the analyzed proteins are exclusively from parasitic origin, and not a consequence of host cell contamination. Flow cytometry is a fluorescence-based technique used to evaluate the expression of extra-and intracellular proteins in different type cells, including protozoan parasites. It also allows the isolation and recovery of single-cell populations. Here, we describe a method to isolate and obtain purified helminthes cells.

[Biological properties of the isolate of Trichinella spp. from a jackal in the North-Caucasian Region].

The biological properties of the isolate from Trichinella from ajackal in the North-Caucasian Region of the Russian Federation were studied. The jackal's muscle tissue showed two Trichinella species preserving their genetic isolation during 5 passages on mice. Oval capsules containing live larvae (on day 90 after infection) in the rat muscles corresponds to the conventional description of the species Trichinella spiralis in their morphometric and biological properties. The morphological data, biological properties, and poor adaptation of round capsule-enclosed parasites to rats indirectly show their affiliation to the other Trichinella species--T. native or T. britovi. There was a negative test for outbred albino rat muscle Trichinella resistance to freezing, which, might be associated with the poor adaptation of this Trichinella isolate to this species of rodents.

Trichinella pseudospiralis in pig from Croatia.

Trichinella spiralis and Trichinella britovi are species that are frequently found in domestic pigs and various sylvatic animals in Croatia. During routine trichinoscopy, non-encapsulated larvae were detected in the muscle tissue of a domestic pig. Artificial digestion revealed a larvae burden of 602 muscle larvae per gram of tissue. Tissue section analysis confirmed the presence of non-encapsulated larvae. Multiplex PCR identified the larvae as T. pseudospiralis. This observation is consistent with the reports of a local veterinary inspector who described the presence of non-encapsulated Trichinella in four individual cases over the last 2 years. This is the first report of T. pseudospiralis in Croatia and one of very few cases of T. pseudospiralis infection described in domestic pigs. The detection of non-encapsulated larvae stresses the need for implementation of artificial digestion instead of trichinoscopy for the detection and identification of Trichinella infections.

Nurse cell of Trichinella spp. as a model of long-term cell cycle arrest.

Nurse cell (NC), formed from skeletal muscle cells upon infection with parasitic nematode trichina, presents a rare system of long-term suspension in the cell cycle. Signaling pathways and general biological functions of Trichinella spiralis NC, inferred from network analysis of competitive expression microarray data (NC vs. C2C12 myoblasts and myotubes), performed in Ingenuity Pathways Analysis (IPA) software and confirmed by Real-Time PCR, are presented. Assuming 4N DNA content in NC nuclei, its cell cycle arrest is identified herein as a hypermitogenic of G(1)-like type, accompanied by induction of senescence, underpinned by increased expression of p15, p16 and p57 cell cycle inhibitors, as well as overexpression of senescence-associated, beta-galactosidase and numerous secretory factors. Growth factor signaling, with predominant role of EGF, cytokine signaling and G-protein-coupled receptor signaling, are suggested as dominant NC signal transduction pathways. Fos, FosB, STAT6, CREBL2, ID4 and retinoic acid dependent nuclear receptors appear to be the main factors determining NC specific gene transcription. Antigen presentation, complement signaling and beta-amyloid processing pathways are also identified as operating in NC. In general, NC pathology is found to pertain to cancer, as well as other, including immunological and neurological, disorders.

Monoclonal antibodies raised in Btk(xid) mice reveal new antigenic relationships and molecular interactions among gp53 and other Trichinella glycoproteins.

Tyvelose-bearing glycoproteins or Trichinella spiralis Group 1 antigens (TSL-1 antigens) are thought to be key molecules in the immunobiology of Trichinella. In the present study, we investigated the binding characteristics of several mAbs produced in Btk(xid) immunodeficient mice that recognise gp53 and some other minor glycoproteins of this parasite. The data obtained reveal the existence of an O-glycan/peptide epitope (recognised by mAb US8) common to all TSL-1 glycoproteins, as well as a specific interaction between the TSL-1 antigen gp53 and other unknown Trichinella glycoproteins in the 35-40 kDa range (these latter react with mAbs US8 and US9, but not with mAb US5). Some of the epitopes recognised by our mAbs are differentially expressed in Trichinella species: the epitope recognised by mAb US5 on gp53 (another O-glycan/peptide epitope) is present only in T. spiralis, whereas those recognised by mAbs US8 and US9 (peptide epitopes) are present in encapsulated Trichinella species. The data obtained also reveal that gp53 is synthesised and glycosylated in beta-stichocytes only. The possible relevance of these findings is discussed.

[The apoptosis in the course of experimental infection with T. pseudospiralis in mice]. / Proces apoptozy w przebiegu doswiadczalnego zarazenia T. pseudospiralis u myszy.

The apoptosis in the course of experimental infection with T. pseudospiralis in mice. The level of apoptic cells in the lamina propria of the mucosa of the jejunum and in the masseter muscle in the mice infected with 200 larvae T. pseudospiralis was observed. Cryostat preparations made from jejunum and muscle samples on 7, 14, 21, 28, 60, 180 and 360 day post infection (dpi), were examined employing the TUNNEL method with the use of "In situ Cell Death Detection KIT Fluorescein" of Roche. The number of apoptic cells in the lamina propria of jejunum started to increase as early as on 7 dpi. The highest number of the apoptic cells, on an average - 56,4 in the crypta-villus unit, was recorded on 28 dpi, whereas the return to the standard level took place only 360 dpi. The level of the apoptic cells in the masseter muscle was the highest on 28 and 60 dpi, which constituted some 30% of all the cells of infiltration.

Remembrance of past images of Trichinella.

The development of achromatic microscopy and the invention of photography were contemporaneous with the earliest investigations on trichinellosis. The former was more important than the latter to 19th century studies on Trichinella. A selection of images, diverse but not comprehensive, is presented to illustrate the early history of trichinellosis.

History of trichinellosis surveillance.

The origin of trichinellosis, which existed in ancient times as testified by the discovery of parasite larvae on an Egyptian mummy, unfolded in several stages: discovery of encapsulated larvae (in the 1820s), identification and scientific description of these larvae (Paget & Owen, 1835), followed by experimental infestations of animals (dogs, pigs, rabbits, mice) or of humans as from 1850. The main occurrences of trichinellosis were followed with particular attention in Europe (Germany, Denmark, France, etc.) and in the United States of America at the end of the XIXth century. They affected numerous domestic animal species (pigs, horses, etc.) or wildlife and humans. Germany paid the heaviest toll with regard to the disease in humans, between 1860 and 1880, with several thousands of patients and more than 500 deaths. Different trichinellosis surveillance systems were set up in the relevant countries in the 1860s. In humans, this surveillance was carried out on affected living patients by a biopsy of the biceps muscles and subsequently by an analysis of eosinophilia (1895). In animals, surveillance was for a long time solely based on postmortem examination of the muscles of the affected animals. This method was used for the first time in 1863 in Germany, and from the 1890s, on several hundreds of thousands of pigs in Europe or in the United States of America.

An immunocytochemical analysis of a class-specific antibody response against Trichinella spiralis in humans.

The response of different classes of antibodies against antigens of the muscle larvae of Trichinella spiralis was tested using an immunocytochemical approach. Ultrathin sections of resin-embedded larvae were treated with sera from patients with trichinosis, then exposed to a biotinated second antibody and stained with avidin-gold complex. Antibody of the M-class was a major component in the response against a slow-responding group of antigens that included stichocyte granules, the cuticle surface, and the esophagus-occupying substance; a minor component in the response against antigens of the rapid-responding group that included cuticle inner layers, hypodermis, hemolymph, and intestinal gland granules. The response of G-class antibody against the rapid-responding group of antigens was detected in all patients tested, while against the slow-responding group of antigens it was detected in only half of the patients, suggesting that an antibody shift from the M to the G class occurred in some patients. The results, obtained in humans, were similar to those we obtained previously in rats (J. Parasitology, 76,230-239, 1990), suggesting that the rat immune system can serve as an experimental model of human trichinosis.

Characterization of a Trichinella isolate from polar bear.

An isolate of Trichinella of polar bear origin was studied by isoenzymatic typing. It was found referable to Trichinella nativa. While the Wistar rats proved nearly refractory to this isolate, the Swiss albino mice were highly susceptible. Ninety-one per cent of the cystic lesions in the diaphragm of the polar bear contained viable larvae after over 20 years of acquisition of the infection by the host which is a case of extreme adaptability of the parasite to its host. The anatomo-pathological aspects of these lesions are studied and the zoonotic significance of this isolate examined.

The intestinal gland of Trichinella spiralis with emphasis on morphology and antigenicity.

The intestinal gland of muscle larvae and adult worms of Trichinella spiralis was investigated with emphasis on its morphology and antigenicity. The gland is situated at the junction between the stichosome and the ampullar portion of the midgut. The cytoplasm was characterized by the presence of cytoplasmic granules. The granules, measuring 1 micron at maximum, were round or sometimes irregular in shape, and of homogeneous appearance with medium to high electron density. Rough endoplasmic reticulum was not prominent. Glycogen granules were absent. The gland was surrounded externally by the basal lamina and hemolymph. The cell membrane was extensively invaginated, and coated pits and vesicles were often observed. The cytoplasm was rather eosinophilic, PAS-negative, and stained red or yellow by AZAN. The cytoplasmic granules were antigenic against trichinosis sera from humans and rats, and cross-reacted with sera from paragonimiasis, trichuriasis and gnathostomiasis patients.

Regional specialization of the surface of a parasitic nematode.

A monoclonal antibody (NIM-M7) has been prepared which reacts with a major surface antigen of adult males and females of Trichinella spiralis. This specificity is only demonstrable when the antigen is liberated by detergent solubilization of surface-labelled worms. When reacted with living adults, on the other hand, NIM-M7 reacts well with only the eversible cloaca, or copulatory bell, of the male, binding weakly, if at all, to other surface areas of male or female worms. A similar staining pattern is also given by Concanavalin A. The differential staining given by NIM-M7 must indicate a molecular difference between the organization of the same surface antigen on the cuticular surface of the copulatory bell and other areas of the worm surface. This example of regional specialization demonstrates that the nematode cuticle is not necessarily chemically uniform.

Treatment of trichinosis with mebendazole.

Prolonged oral high-dose mebendazole therapy provided an effective anthelmintic regimen for trichinosis unresponsive to steroid therapy in one patient. Side effects were limited to a Herxheimer-like reaction. Serum mebendazole levels documented gastrointestinal absorption. Repeat muscle biopsies and fluorescein angiography substantiated objective improvement.

[Feeding behavior of Trichinella as a leading factor of adaptation to the body of the mammals]. / Sposob pitaniia Trikhinelly kak vedushchii faktor adaptatsii k organizmu mlekopitaiushchikh

Studies were carried out of the permeability of the cuticle of Trichinella spiralis (Owen, 1835) for fluorochromes and histidine, the filling of the mid-gut and the amino-acids contents in it. The quantity of glicogene in different organs was estimated. A conclusion has been grown that a larva consumes food through the integuments of its body; intestinal Trichinella use within the first 20 hours of their development the reserves of glicogene and later consume food monomeres and oxygen from the host's mucous membrane through their integuments. The mode of feeding of Trichinella was formed during their evolution as an adaptation to parasitism on the mucous membrane of the host.

On the karyotype of a laboratory Trichinella strain from Bulgaria.

The karyotype of male and female individuals of the species Trichinella nelsoni was studied. It was found that the number of chromosomes in females individuals is 2n = 6 and in males 2n = 5. Each pair of chromosomes differs from one another as to dimensions and location of the centromere. The univalent chromosome that was found in the chromosome set containing five chromosomes is the second largest submetacentric chromosome. It is suggested that this chromosome is the sex chromome of the studied Trichinellae.