The Pathogenesis Of Simian Immunodeficiency Virus Infection And The Modulation Of Tradional Chinese Medicie

The process by which HTV progresses to AIDS occurs over a long and slow time period during which there is a continual and complex interaction between HIV and the immune system. A multitude of causes have been raised to explain its pathological mechanism, including the theory that HTV directly kills immune cells. Various pathogens, including fungus, virus and bacteria infection have an effect on the proliferation activity of HIV and the interactivity of CD4 with gp120. This produces a negative signal leading to injury of CD4+ cell function. Because the membrane proteins of HIV and the MHCâ…¡ antigens share a homogenous sequence, autoimmune anti-MHC â…¡ antigens inhibit the immunological functions of the CD4+ cells. Recently experts have posited various theories, including cellular autolysis, mutations of cell factors from Th1 type to Th2 type, as well as autoimmune pathology caused by unneeded energy provided by process gp120 antigens to already active CD4+ cells causing CD4+ cell cytotoxicity.Numerous theories exist regarding the mechanism of HIV disease progression. Possibly the etiological changes of HIV infection have important effects on its progression and development. However, it is still difficult to ascertain with any certainty the main factor(s) responsible for the ultimate development of AIDS. Looking at the problem from a total perspective, the above theories all are likely superficial events. The root of the problem probably is linked to immunological activation. The immune system is never able to eradicate completely HIV infection. The viruses persistent existence leads to various types of chronic effector cell activation by the immune system. Under certain conditions, this continual activation of the immune system is beneficial in that it inhibits HIV proliferation and stops the spread of HIV. Yet, other conditions can cause over-activation destroying normal immunoregulation. This leads to immunological failure which harms the body and promotes disease progression. From this point of view, all types of immunological activation, regardless whether at the original acute infection time or during the chronic latency period, have a double-sided effect. It both protects and harms the body. For example, to elicit special immune responses it is extremelyimportant that HIV is held in the lymphatic tissue right from the start. As such, because HIV can directly infect the immune system cells, the lymphatic tissue also become excellent grounds for establishing initial infection. This, along with immunological activation related to primary specific immunological response, are normal links within a comprehensive immune response. However, activated immune cells also aid HIV proliferation and promote their spread.Complement fixation antibodies produced during primary infection assist the dendritic cells expressed from within the center of lymph nodes capture HIV and eradicate the viruses. However, these captured viruses can also create new infection. Regarding HTV induced changes in cell factor secretion, some cell factors, such as IFN-a, are able to inhibit HIV proliferation and strengthen immune response while others will promote HIV proliferation or inhibit immune response. The perpetual existence of the virus can stimulate the body to continually produce protective immunological responses. However, certain viral antigens, like gp 120, are able to emit negative signals against CD4+ cells leading to 'no response ' and thereby induce homologic antigen response or produce anti-MHCII type antigen antibodies and inhibit T cell functioning. Under normal conditions, cell autolysis can limit the activated effector cells. During HTV infection, however, there is an over abundance of activated effector cells producing large amounts of cell autolysis which damage the immune system. HTV specific cytotoxic T cells have an important role in inhibiting and eradicating HIV. Yet, they also can exhaust antigen expressing cells and create a pathological immune response. Ideally one must appropriately induce while also inhibit immunological activation; wield its benefits while avoiding its negative side effects. Balancing this type of yin-yang relationship serves to allow the immune response to inhibit and eradicate HTV without causing excessive, harmful side effects. This is truly the ultimate method by which the core presence of AIDS can be prevented, controlled, cured and completely eradicated.Epimedium Brevicormon Maxim is a traditional Chinese medical herb used to tonify the kidneys. While its pharmacological activity and effective mechanisms have been well studied, there is still no report on its anti-viral activity. This project uses the SIVmac239 -CEMxl74 system to prove the anti-SIV replication effects of one of Epimedium Brevicormon Maxim's main ingredients, Ic-ariin. Results demonstrate that at subacute concentrations, Ic-ariin has an excellent dose-dependent effect in inhibiting SIV replication in vitro. Its TC50 is 74ug/ml with an IC50 of 36ug/ml. Fundamentally this project, advances the research concerning the mechanism by which Ic-ariin inhibits SIV replication with regard to the relationship between the post-infection viruses and thehost cells with particular attention to the mechanism by which the virus induces host cell apoptosis.To determine whether the changes in apoptosis and intracellular signaling transduction could be attributed to viral infection, we examined samples of control and SIVmac239 infected CEM xl74 cells by microscopy and PCR analysis. Syncytia formation could be observed after 24 h incubation with SIVmac239. SIVmac239 DNA could be detected after 12 h of infection. The result shows that a 381 bp fragment of the SIV gag gene was detected only in the CEM xl74 cells treated with the virus proving that the SIV successfully infected the CEM xl74 cells.The viability of normal (control) and infected CEM xl74 cells was determined by MTT assay. There was a pronounced decrease in cell viability in the SIVmac239 infected group with no Ic-ariin compared to the control cells. After treatment with different concentrations of Ic-ariin, viability in the infected group was fundamentally restored. These results showed that Ic-ariin's TC50 is 74ug/ml and its IC50 is 36ug/ml. At subacute concentrations, Ic-ariin demonstrated an excellent dose-dependant relationship against SIV replication. Immonoflorescence indicated that 74ug/ml of Ic-ariin treated cell antigens had a positive inhibition rate of 100%. 36ug/ml of Ic-ariin had an inhibition rate of 89.4% and 18ug/ml reached 43%. These numbers demonstrate that Ic-ariin has an excellent, though dose dependant, inhibitory effect on SIV replication.Loss of plasma membrane asymmetry is an early event in apoptosis and results in the exposure of phosphatidylserine (PS) residues at the outer plasma membrane leaflet. Annexin V, a phospholipid binding protein, specifically binds to PS. Annexin V binding assays showed that most of the viable cells in both groups were annexin V-negative, but a higher proportion of apoptotic cells appeared in SIVmac239 infected cells. Ic-ariin increased the percentage of viable cells while also decreasing the percentage of apoptotic cells in both the normal and SIVmac239 infected groups. ANOVA analysis showed that the effect of Ic-ariin in the SIVmac239 infected group was more marked than in normal cells (P<0.05).The influence of Ic-ariin on intracellular cAMP concentration 15 min post-infection was determined. Ic-ariin at 50(Ag/ml reduced the concentration of cAMP in both normal and infected groups, but the effect was more marked in the latter (ANOVA analysis P<0.01). Ic-ariin at 50 [Ag/ml down regulated PKA activity in both normal and infected groups, but again was more marked in the latter (ANOVA analysis P<0.05).Levels of PKA-dependent histone phosphorylation were detected by Westernblotting. The effect of Ic-ariin at 50 fig/ml was seen after 2 h of treatment (comparing time points between 0.5 h and 8 h).The result shows that phosphorylated histone H3 was elevated after viral infection and significantly decreased in both normal and SIVmac239 infected groups in the presence of Ic-ariin which, again, was more marked in the latter.It has been reported that cAMP-PKA is translocated from the golgi area to the nucleus. Apoptosis may be mediated by PKA through histone H3 phosphorylation. It has also been verified that histone H3 can be phosphorylated on serine-10 via the cAMP-PKA signal transduction pathway. The over expression of cAMP-dependent PKA could lead to massive serine-10 phosphorylation and block the cells' entry into M phase. The data in this study shows that the level of phosphorylated histone H3 is elevated in SIVmac239 infected CEM xl74 cells but is suppressed by Ic-ariin in both normal and infected groups.These results suggest that apoptosis triggered by SIVmac239 infection is a consequence of the cAMP-PKA signal transduction cascade. Short-term exposure of SIVmac239 infected CEM xl74 cells to Ic-ariin may temporarily alleviate the progression of apoptosis induced by SIVmac239, but the longer survival of infected cells will eventually favor replication and production of viral particles and eventually lead to cell death.A self-cleaving hammerhead ribozyme was designed by introducing a fragment of ribozyme target sequence downstream from the catalytic sequence. A self-cleavage ribozyme gene was synthesized, amplified and cloned at the Xhol-Sall site of Bluescript SK. The construct, bearing 10 copies of ribozyme, was attained through successively cloning four times. The result from activity assays in vitro showed that self-cleaving ribozymes possess quite high activity in cis- or trans-cleavage reaction.The monomer ribozyme gene and the 10-mer ribozyme constructed on pBluescript SK were transferred into the expression vector pCI-neo. The expression vector harboring ribozyme gene transfected lymphocyte CEMx-174 by liposome-mediated transfection. The cell clones with stable growth were obtained after selection in the medium containing 400 n g/mL. Northern dot blot results indicated that the monomer ribozyme gene and the 10-mer ribozyme gene were transfected and expressed in the cells. After culturing one month, the transfected cells were inoculated with simian immunodeficiency virus stock 10TdD50. The pathological changes of the cells were observed under the upside-down microscope. Observations show that the cells transfected with the monomer ribozyme gene grew normally,were not observed the cytopathic effect(CPE),that the most of the cells transfected with the 10-mer ribozyme...