Assessment of the In Vitro and In Vivo Antitumor Activity of Hemocyanins from Helix aspersa, Helix lucorum, and Rapana venosa in a Graffi Myeloid Tumor Model.

Hemocyanins are oxygen-transporting glycoproteins in the hemolymph of some invertebrate species that attracted scientific interest as potential anticancer agents. The present study aims to assess the in vitro and in vivo anticancer activity of hemocyanins isolated from Helix aspersa, Helix lucorum, and Rapana venosa in the Graffi myeloid tumor model. The in vitro antitumor activity of the hemocyanins was determined by a MTT test and cytomorphological analysis by fluorescent and transmission electron microscopy. The in vivo effects of the hemocyanins were examined in hamsters transplanted with Graffi tumor. The serum antibody titers against the tested hemocyanins and tumor antigen were determined by ELISA. Histopathological assessment of the morphological features related to antitumor effect, immune system response, and toxicity in some internal organs was performed. The results of in vitro studies indicated that the tested hemocyanins induced significant antiproliferative and apoptogenic effects. The in vivo investigations demonstrated a protective antitumor effect, expressed in reduced transplantability, suppression of tumor growth and metastasis, reduced mortality, prolonged survival time, and absence of toxic side effects. The present study indicated that the antitumor activity of the studied hemocyanins was due to both immune stimulation and direct effects on the tumor cells, and they displayed their potential as therapeutic agents against hematological malignances.

Trichinella spiralis (Owen, 1835) Induces Increased Dystrophin Expression in Invaded Cross-striated Muscle.

PURPOSE: Dystrophin and the dystrophin glycoprotein complex serve as a cytoskeletal integrator, critical for muscle membrane stability. The aim of the present study was to clarify the expression of dystrophin protein and mRNA in the skeletal muscle tissue during the muscle phase of trichinellosis in mice. METHODS: Muscle tissue was collected from mice experimentally infected with Trichinella spiralis at days 0, 14 and 40 after infection. The expression of dystrophin in the muscle tissue was investigated by immunohistochemistry with antibodies against three different domains of the protein, and the expression levels of Dys mRNA by real-time PCR. RESULTS: The presence of dystrophin protein was increased in the de-differentiating cytoplasm at the early stage of muscle infection and was persisting also in the mature Nurse cell harbouring the parasite. It was accompanied by significantly elevated expression of Dys mRNA at days 14 and 40 after infection. CONCLUSION: Our findings indicate that dystrophin plays a role in regeneration of the muscle and in the Nurse cell formation and stability for security of the parasite survival.

Hemocyanins from Helix and Rapana Snails Exhibit in Vitro Antitumor Effects in Human Colorectal Adenocarcinoma.

Hemocyanins are oxygen-transporting glycoproteins in the hemolymph of arthropods and mollusks that attract scientific interest with their diverse biological activities and potential applications in pharmacy and medicine. The aim of the present study was to assess the in vitro antitumor activity of hemocyanins isolated from marine snail Rapana venosa (RvH) and garden snails Helix lucorum (HlH) and Helix aspersa (HaH), as well the mucus of H. aspersa snails, in the HT-29 human colorectal carcinoma cell line. The effects of the hemocyanins on the cell viability and proliferation were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the alterations in the tumor cell morphology were examined by fluorescent and transmission electron microscopy. The results of the MTT assay showed that the mucus and α-subunit of hemocyanin from the snail H. aspersa had the most significant antiproliferative activity of the tested samples. Cytomorphological analysis revealed that the observed antitumor effects were associated with induction of apoptosis in the tumor cells. The presented data indicate that hemocyanins and mucus from H. aspersa have an antineoplastic activity and potential for development of novel therapeutics for treatment of colorectal carcinoma.

Clinical and epidemiological descriptions from trichinellosis outbreaks in Bulgaria.

Bulgaria is one of European countries where trichinellosis continues to be regularly diagnosed and registered. The clinical and epidemiological features of 72 cases of trichinellosis associated with five outbreaks caused by Trichinella spiralis and Trichinella britovi between 2009 and 2011, are described. At hospital admission, patients were often initially treated with antibiotics, without any improvement. A range of signs and symptoms were recorded, including: myalgia, elevated temperature, arthralgia, difficulty with movement, facial oedema, conjunctival hyperaemia, ocular haemorrhages, diarrhoea, skin rash, headache, and fatigue. Due to the variable clinical course of the disease, the diagnostic process for trichinellosis is often complex and difficult. This means the diagnosis may be established late for an appropriate treatment, potentially leading to a severe course of the disease with complications. Laboratory abnormalities were expressed by marked eosinophilia (97.2%), leucocytosis (70.8%), elevated serum creatine phosphokinase levels (82%), and antibody-positive results by ELISA and indirect hemagglutination. Patients were treated with albendazole (Zentel) 10 mg/kg for 7-10 days. In two outbreaks, the aetiological agent was T. spiralis, in one outbreak T. britovi, and an unknown Trichinella species in the fourth outbreak. The sources of infection were domestic pigs, probably fed with scraps and offal of wild game. In one outbreak, T. spiralis was also detected in brown rats trapped close to where the pig had been raised in the backyard. These epidemiological factors are relevant in considering implementation of targeted control programmes.

Accumulation of α-2,6-sialyoglycoproteins in the Muscle Sarcoplasm Due to Trichinella Sp. Invasion.

The sialylation of the glycoproteins in skeletal muscle tissue is not well investigated, even though the essential role of the sialic acids for the proper muscular function has been proven by many researchers. The invasion of the parasitic nematode Trichinella spiralis in the muscles with subsequent formation of Nurse cell-parasite complex initiates increased accumulation of sialylated glycoproteins within the affected area of the muscle fiber. The aim of this study is to describe some details of the α-2,6-sialylation in invaded muscle cells. Asynchronous invasion with infectious T. spiralis larvae was experimentally induced in mice. The areas of the occupied sarcoplasm were reactive towards α-2,6-sialic acid specific Sambucus nigra agglutinin during the whole process of transformation to a Nurse cell.The cytoplasm of the developing Nurse cell reacted with Helix pomatia agglutinin, Arachis hypogea agglutinin and Vicia villosa lectin-B4 after neuraminidase pretreatment.Up-regulation of the enzyme ST6GalNAc1 and down-regulation of the enzyme ST6GalNAc3 were detected throughout the course of this study. The results from our study assumed accumulation of sialyl-Tn-Ag, 6`-sialyl lactosamine, SiA-α-2,6-Gal-ß-1,3-GalNAc-α-Ser/Thr and Gal-ß-1,3-GalNAc(SiA-α-2,6-)-α-1-Ser/Thr oligosaccharide structures into the occupied sarcoplasm. Further investigations in this domain will develop the understanding about the amazing adaptive capabilities of skeletal muscle tissue.

Increased sialylation as a phenomenon in accommodation of the parasitic nematode Trichinella spiralis (Owen, 1835) in skeletal muscle fibres.

The biology of sialic acids has been an object of interest in many models of acquired and inherited skeletal muscle pathology. The present study focuses on the sialylation changes in mouse skeletal muscle after invasion by the parasitic nematode Trichinella spiralis (Owen, 1835). Asynchronous infection with T. spiralis was induced in mice that were sacrificed at different time points of the muscle phase of the disease. The amounts of free sialic acid, sialylated glycoproteins and total sialyltransferase activity were quantified. Histochemistry with lectins specific for sialic acid was performed in order to localise distribution of sialylated glycoconjugates and to clarify the type of linkage of the sialic acid residues on the carbohydrate chains. Elevated intracellular accumulation of α-2,3- and α-2,6-sialylated glycoconjugates was found only within the affected sarcoplasm of muscle fibres invaded by the parasite. The levels of free and protein-bound sialic acid were increased and the total sialyltransferase activity was also elevated in the skeletal muscle tissue of animals with trichinellosis. We suggest that the biological significance of this phenomenon might be associated with securing integrity of the newly formed nurse cell within the surrounding healthy skeletal muscle tissue. The increased sialylation might inhibit the affected muscle cell contractility through decreased membrane ion gating, helping the parasite accommodation process.

The occupation of intestinal epithelium by Trichinella spiralis in BALB/C mice is not associated with local manifestation of apoptosis related factors.

Trichinella spiralis actively passes through the epithelial cells of the intestinal mucosa but morphologically, these cells do not manifest apparent damage. The possible activation of apoptotic mechanisms in the small intestine mucosa after infection with larvae and adults of Trichinella spiralis was explored by immunohistochemistry. Sporadic individual cells of normal intestinal epithelium showed activation of caspase-3, increased expression AIF, or Bax. The larval stage of intestinal trichinellosis was characterized by distortion of cells on the villus tips that were strongly reactive to caspase-3, Bax, and survivin antibodies. There was a transient loss of the survivin expression on the brush border of the epithelial cells at 15-h post infection, which reappeared on the fifth day. Bcl-2 changed its normal apical distribution and re-localized to the basal part of the epithelial cells. No significant changes of expression of the selected apoptosis-related proteins were observed in the intestinal epithelial cells immediately surrounding the worms. The presence of Trichinella affects intestinal epithelial cells, but unlike in muscle cells, invading them does not initiate apoptotic factors activation.

Apoptosis as the adaptation mechanism in survival of Trichinella spiralis in the host.

The study evaluates the role of apoptosis-inducing factor (AIF) in the process of striated muscle cell transformation after occupation by Trichinella spiralis. Its relationship with other apoptosis-related factors [apoptotic protease-activating factor 1, Bcl-2 associated protein X (BAX), Bcl-2, caspase 3, survivin, poly (ADP-ribose) polymerase-1 (PARP-1), and endothelial and inducible (iNOS) nitric oxide synthase] was evaluated by immunohistochemistry. In the context of low BAX and caspase 3 expression and strong distribution of AIF in the sarcoplasm and nucleus at the very early stage of infection, we suppose that AIF-mediated signaling is involved in the apoptosis activation in the area of Trichinella occupation. In the time course of nurse cell formation, survivin and caspase 3 migrated into the enlarged nuclei with strong PARP-1 expression. In the end of encapsulation of Trichinella, expression of all proapoptotic factors ceased and only survivin in the nuclei and Bcl-2 positivity in the cytoplasm persisted in the formed nurse cell. The expression of sarcoplasmic iNOS was absent during the process of muscle cell de-differentiation and reappeared within the nurse cell. It seems that upregulation and downregulation of factors of apoptosis in the skeletal muscle cell represents an adaptive mechanism providing a comfortable niche for the parasite.

Selenium supplementation at low doses contributes to the antioxidant status in Trichinella spiralis-infected rats.

Selenium (Se) supplementation may prevent the formation of free radicals and lipid peroxidation processes in trichinellosis. The oxidative-antioxidant status of male Wistar rats infected or uninfected with Trichinella spiralis (Nematoda) and supplemented or unsupplemented with Sel-plex (Alltech) was tested through blood biomarkers. Sel-plex was applied to restore antioxidant defense system. The oxidative marker was malondialdehyde (MDA) concentration. The antioxidant markers were superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities and concentrations of Se and vitamin E. The animals were allocated into four groups. The experiment covered 8 weeks post infection. A mathematical model was proposed for the time course of host body weight. The model solutions were in good agreement with the experimental data. The relative rates of body weight gain were determined as growth kinetic parameters. The supplementation of the rats with dietary Se improved their antioxidant status. Increases by 10% in SOD activity, 6% in GPx activity, 13% in vitamin E concentration, 17% in plasma Se concentration, and 19% in liver Se concentration, respectively, and a decrease by 18% in serum MDA concentration were recorded in the infected and supplemented towards infected and unsupplemented rats. The reduction of muscle larvae after Sel-plex application was 63%. The mortality in infected and uninfected animals did not differ significantly. No statistically significant differences were established between the growth of the control and infected rats. At week 8, the body weight gain in the supplemented rats (both uninfected and infected) was 30% higher, compared to that in unsupplemented ones. Diet with Se could be beneficial in the treatment of diseases correlated with considerable oxidative stress, particularly parasitoses.

Miltefosine decreases the cytotoxic effect of epirubicine and cyclophosphamide on mouse spermatogenic, thymic and bone marrow cells.

A new class of potent anticancer drugs, alkylphosphocholines has been recognized lately. Miltefosine (Hexadecylphosphochlorine, HPC) has been found to express select antineoplastic effect on human breast cancer skin metastases with simultaneous preservation of bone marrow proliferative activity and low clastogenicity. In the current study, we present data about the specific effect of two widely used cytostatics Cyclophosphamide (CP) and Epirubicine (ERb) applied separately or in combination with Miltefosine. C57BL6 mice were treated per os or intraperitonieally in doses corresponding to that in clinical use. Morphological, autoradiographic, ultrastructural and cytogenetic studies on spermatogenic, thymic and bone marrow cells were performed. It is found that compared with separate application, combinations of ERb or CP with Miltefosine slightly decreases spermatogonial proliferation and exerts milder effect on the structure of germinal and thymic cells. In addition, a lot of plasmocytes showed signs of active protein (antibody) synthesis. A significant reduction of aberrant chromosomes (clastogenicity) without changes in proliferative activity of bone marrow cells were recorded. In conclusion, the combine application of Miltefosine with ERb and CP decreased the destructive cytotoxic effects of ERb and CP on mouse spermatogenic and hematopoietic cells.