Analyzing Trichinella Spires Infection - Essay Example

Introduction Trichinella is a roundworm found in many carnivorous animals. The roundworm is well distributed globally making it a successful parasite. The parasite is of class adenophora, subclass enoplia. The parasite belongs to the order stichosomid and the family of trichenrllidae.The Trichinella parasite was first described by Richard Owen and James Paget. Trichinella can cause infections in humans that can lead to mortality and serious morbidity in humans. The species has been categorized into eleven displaying diverse biological characteristics that pose a threat to human health and wellbeing. Morphology Males and Females taper at the anterior part. The males are 1.3- 1.6mm long whereas the females are double the size of the males. The infective larvae are coiled into a spiral. Picture of an adult male courtesy of Trichinella.com The posterior end of a female contains a uterus full of eggs. Pictures courtesy of Trichinella.com. Picture of larvae (Villella 1970) Life cycle of Trichinella The life cycle of Trichinella begins with larvae being ingested raw or undercooked, pork meat. The larvae attach and bury themselves in the intestinal mucosa developing into adults in the small intestines. The adults in the intestinal mucosa mate producing more larvae. A single female can produce up to 1500 over a period of 1 to for weeks. The produced larvae then enter the lymph and mesenteric veins traveling throughout the body eventually attaching itself as a cyst in the striated muscle. The cyst helps the Trichinella species to survive the host’s immune system. The larva may remain viable for up to 25 years in humans. The viable cysts when ingested by a new host they become viable again beginning the cycle. The nematodes of genus Trichinella infect a wide range of mammal’s reptiles and birds. The parasite usually alternate between the enteric and skeletal muscle stages within their hosts. The eleven known genotypes are made up of two categories. The first is the species that encapsulation occurs and that one that no encapsulation occurs. (Zarlenga et al. 2004). T spirally is the most common cause of human infections, and much of the proposal will discuss this species. The T spirally usually inhabits the host muscle cells and become encapsulated. The Trichinella species is found in a broad geographic range from the tropics to the Arctic. The encapsulated species shows more adaptations to colder climates as compared to the encapsulated species. The major hosts of Trichinella species are sylvatic swine and domestic swine. The synanthropic group of animals such as rat’s cats, dogs, and carnivore also from the host to the Trichinella species. The high infections to swine mean that Trichinella species spread to most continents thus showing a cosmopolitan distribution in equatorial and temperate climates (Pozio 2001). The species has been classified into three by use of molecular phylogenetics(Blaxter et al. 1998). Dorylaimia, Enoplia, and Chromadorea(including Tylenchina-Clade IV, Spirulina-Clade III, and Rhabditina-Clade V).Nematodes and Arthropods diverged 700 – 1000 million years ago Blaxter 1998).The most recent common ancestor of C. Elegans, Trichostrongylus Nippostrongylus brasiliensis (Clade V), colubriformis (Clade V), Pseudoterranova decipiens (Clade III) and ascaris suum(Clade III) lived about 550 Million years ago. A comprehensive and robust analysis of the biogeography and phylogeny history of Trichinella species has been recently reported based on the genetic variations of different species (Zarlenga et al. 2006). Trichinella results in the formation of a capsule in the infected muscles. The nurse CELL is composed of the fibrous wall and cellular components. The nurse cell formation is a complex process that includes cell cycle arrest reentry and differentiation. Parasites alter the morphological characteristics of hosts to survive in the hosts bodies. The Trichinella parasite makes a new architecture in the host tissue. The capsule built by Trichinella is made up of cellular components and a collagenous wall. The cellular component enables parasite metabolism while wall offers protection. The cellular components are referred to as the nurse cell since it is made up from the host’s biological resources. The prominence of the capsule in the infected muscle is seen even by armature pathologist. The construction of a unique and favorable living space for Trichinella is an interest to scientists. Maier and Zaiman espoused the similarities between changes that occur during nurse cell formation and muscle regeneration. Read, and Steward presented detailed comparison of the two processes using ultra structural and biochemical processes that occur. Their analysis was that the process of regeneration plays an important role in the nurse cell development. Trichinella uses the repairing process of muscles to construct the capsule when the parasites induce injury by invasion. The muscle cells undergo a process of repair like in any other trauma. Trichinella borrows initial part of the process of repair to building its home. A skeletal muscle cell is prone to injury by direct trauma and or indirect causes. The skeletal muscle has remarkable ability regeneration. The regeneration process involves four processes, cell activation, cell proliferation, differential on and fusion. The invasion by Trichinella causes the muscle cell to damage thus initiating activation of the satellite cells which undergo proliferation and redifferentiation. An increase exists in the amount of sarcoplasmic matrix and muscle regeneration during the process. The process is associated with an increase in number of nuclei, RNA, DNA, and sarcoplasmic reticulum. The nurse cell formation process can be best understood by understanding the muscle genesis. The skeletal muscles come from mesoderm cells that originate from the somites the mesodermal precursor cells are specific to myoblasts. As the myoblasts proliferate, they withdraw from the cell cycle terminally differentiating to myocytes. The process of muscles Genesis is fusion of mononucleated mytocyes to form multinucleated cells that eventually forms muscle fibers (Wu et al. 2008). The process of muscle repair following injury are the accumulation of inflammatory cells in the damaged site. Neutrophils and macrophages phagocytes the cellular debris affecting muscle regeneration by activating myogenic cells. The muscle degeneration activates muscle repair process. The satellite cells differentiate and fuse with one another forming new myofibrils. Capsule formation is known as cytogenesis takes a period of 20 days from the time of larval invasion to nurse the cell completion. The infection usually causes profound changes in the host muscle cell. The dissolution and complete loss of myofibrillar then occurs. The infection thus causes activation, proliferation and cell differentiation of the cells. The pathways and genes mobilized by the process are death receptor pathway and mitochondrial pathway. The nurse cell usually developed has as many as 100 nuclei that are enlarged. The nuclei usually possess nucleoli surrounded by a collagenous capsule wall and a circulatory structure. Thethis study aims to determine if the cell cycle proteins are expressed at different stages of nurse cell development in Trichinella spires infected muscle. Material and Methods The methodology employed will be by use of antibodies to determine the cell cycle proteins expressed at the different nurse cell development. Rat muscle infected with T spirallis were dewaxed 10 12, 14, 18, 24, and 32 days post infection. Antibodies were used against cyclin proteins to determine which cylins are expressed in developing nurse cells. Light microscopy was used to determine the number of positive cells at each point and time as compared to different time points. An uninfected muscle tissue was used in the same section as a control. The process of identification was by use of Elisa; the samples of Rat muscle were lysed in coating buffer (0.15 M NaHCO3, pH 9.07 with ultrasound 21 kHz) in an ice bath. The muscle lysates s were centrifuged, and the concentration of muscle protein was determined from the resulting supernatant. This was done using the commercial reagents that established amount of bovine serum albumin as a standard. The microtiter well plates were then coated overnight with 200 mg of crude protein antigen in 100 l of the coating buffer per well at room temperature. Wells were then washed once with saline, and a staining was blocked with buffer (5% wt/vol] skimmed milk and 0.2% Phosphate-buffered saline for 1 hour at room temperature (Woznik et al. 2005). After three washings, the wells were incubated with a serial dilution of test sera in saline and kept in incubation for 1 hour at 37 C. Wells were then washed three times, and 100 l of peroxidase-conjugated goat anti mice (Serotec diluted 1: 10,000) was placed inside the wells for thirty minutes at 37 C (Wu et al. 2008). Wells were then washed four times, and tetramethylbenzidine substrate was added to the reaction. The reaction was ended after approximately 2–4 minutes according to color appearance with 1 N sulfuric acid, and absorbances measured at 450-nm wavelength (Despommier 1998). The cut-off value for positive samples was set at the mean absorbance of the negative animals plus double the SD at the termination of the experiment (Wu et al. 2008). Immunocytochemical localization parts of Trichinella larvae and cysts from mice obtained at 12, 14 18 24 and 32 weeks post infection were incubated for 60 minutes at room temperature in sera (dilution 1: 100) from mice with homologous and heterologous Trichinella species infections. Serum from a control animal served as a negative control and serum were used for blocking. Sections were incubated for thirty minutes with the same secondary antibody used in the ELISA and Western blot, and aminoethyl carbazole serving as the chromogen. Statistics Data are expressed as mean. To analyze data from every tissue cysts, which were put together with experimental animals. The variations between groups at each time of sampling were tested by use of a mixed-model analysis of variance with group as a fixed variable and tissue parts as a random variable nested within the group (Wozniak 2005).The effect of time was analyzed differently in each experimental group, with time as a fixed variable. A one-way t-test and ANOVA were used. Results and Discussion The binding of IGF-I receptor to the IGF-I induces phosphorylation in the receptor, and that acts through MAP-kinase kinase through the MAP kinase pathway. It then activates cell cycle progression genes that proliferate in the satellite cells after the Trichinella infection (Wu et al, 2005). This happens through the PI3-K/Act pathway modulating the expression of muscle differentiation genes which are involved in the redifferentiation of satellite cells and the differentiation of infected muscle cells (Wu et al, 2005). The activation of PI3-K/Akt inhibits the pro apoptosis by cl-2 family and induces anti-apoptotic function by Bcl-2 family that contributes to the survival of nurse cells. This figure referred the review by Mourkioti and Rosenthal (Wu et al, 2008). Following the invasion of newly born larvae, the infected muscle cell withdraws from the G0 cell cycle and re-enters the cell cycle (Wu et al, 2008). The enlarged nuclei possess an approximate complement of DNA. The increased DNA synthesis is then completed by five dpi, and then is changed throughout the course of infection, which indicate that cell cycle has been arrested. The molecular mechanism of cell cycle arrest and reentry during infection remains unknown. However, previous studies have illuminated on the matter (Gross et al, 2005). The cell cycle arrest may be unique to the nurse cell formation since no similar phenomena were reported in muscle repair processes or muscle Genesis, as far as present authors are aware (Jasmer, 2005). The expression change of many cell cycle-related factors was observed in Trichinella infected muscle tissue, retinoblastoma (Rb), CDK4, cyclin B2, cyclin c cyclin D2 and cyclin D3, CLU, G0/G1 switch gene 2 (G0S2),(Wu et al 2005) inhibitor of DNA binding 2 (Id2), myeloblastosis oncogene (Myb), and N-myc downstream regulated gene 2 (Ndrg2. Different cyclins bind allosterically to different CDKs to form distinct complexes at specific phases of the cell cycle and thereby direct the cell from one stage of the cycle to another (Myer & Zerman 1966). Upon stimulation, the cyclins of D-type assemble CDK4 and CDK6 to form complexes, which facilitate cells exit of the G0 phase and re-enterance into the cell cycle of G1 cell cycle phase, Therefore, cyclin D2, cyclin D3, and CDK4 increased expression is probably involved in the cell cycle reentry after infection (Wu et al, 2008). Up-regulated symbol of retinoblastoma, P21, P27 cyclin-dependent kinase inhibitor 1B and p57 could attribute to the cell cycle arrest of infected muscle cell (Parker 2003). These actors are known to play a critical role in the growth arrest of differentiating cells because it specifically inhibits CDKs leadsing to the withdrawal of cells from the cycle and differentiation (Parker 2003). As a cyclin-dependent kinase inhibitor, P21 is critical factors in cell cycle arrest at G2/M (Gross et al, 2005). Cells with little P21 are unable to maintain stability of the cycle arrest. Introduction of non-functional P21 or a P21 antisense oligonucleotide diminished the G2/M arrest phenotype in cells In Trichinella infection, expression of P21 was up-regulated, which increased from 13 dpi, decreased after reaching a peak of 18 dpi at late stage of infection. Therefore, P21 is an important factor in cell cycle arrest during nurse cell formation (Wu et al, 2006). The changes in expression changes of several other cell cycle-related genes were also observed in Trichinella infection, such as CLU and G0S2 (Matsuo et al, 2001). The expression of CLU was up-regulated while the expression of G0S2 was regulated down. It is known that, the two genes play roles in regulating the cell cycle (Zammit et al, 2006). An excessive expression of CLU brought about an increased accumulation of cells at the G0/G1 phases of the cell cycles, accompanied by slowdown of cell cycle progression and a reduction of DNA synthesis High level of CLU causes cell cycle arrest G0S2 is induced transiently upon the cells re-entry into the phase, G1 of the cell cycle (Zammit et al, 2006). Therefore, UCL and G0S2 may be involved in the arrest of infected muscle cells (Wozniak et al, 2005). One of the significant signaling pathways involved in cell cycle arrest is the TGF-β signaling pathway (Wozniak et al, 2005). TGF-β is cytokine that controls cell proliferation, differentiation apoptosis and morphogenesis through a series of sad proteins (Wozniak et al, 2005). The TGF-β is signaling pathway thus causes cells to cease proliferation and to down-regulates the genes that promote cell cycle progression through the S phase, leading to the arrest of the cell cycle (Steward et al, 1973). 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