Evaluation of Leishmania donovani Protein Disulfide Isomerase as a Potential Immunogenic Protein/Vaccine Candidate against Visceral Leishmaniasis

In Leishmania species, Protein disulfide isomerase (PDI) - a redox chaperone, is reported to be involved in its virulence and survival. This protein has also been identified, through proteomics, as a Th1 stimulatory protein in the soluble lysate of a clinical isolate of Leishmania donovani (LdPDI). In the present study, the molecular characterization of LdPDI was carried out and the immunogenicity of recombinant LdPDI (rLdPDI) was assessed by lymphocyte proliferation assay (LTT), nitric oxide (NO) production, estimation of Th1 cytokines (IFN-γ and IL-12) as well as IL-10 in PBMCs of cured/endemic/infected Leishmania patients and cured L. donovani infected hamsters. A significantly higher proliferative response against rLdPDI as well as elevated levels of IFN-γ and IL-12 were observed. The level of IL-10 was found to be highly down regulated in response to rLdPDI. A significant increase in the level of NO production in stimulated hamster macrophages as well as IgG2 antibody and a low level of IgG1 in cured patient's serum was observed. Higher level of IgG2 antibody indicated its Th1 stimulatory potential. The efficacy of pcDNA-LdPDI construct was further evaluated for its prophylactic potential. Vaccination with this construct conferred remarkably good prophylactic efficacy (∼90%) and generated a robust cellular immune response with significant increases in the levels of iNOS transcript as well as TNF-α, IFN-γ and IL-12 cytokines. This was further supported by the high level of IgG2 antibody in vaccinated animals. The in vitro as well as in vivo results thus indicate that LdPDI may be exploited as a potential vaccine candidate against visceral Leishmaniasis (VL)

Proteins of Leishmania (Viannia) shawi confer protection associated with Th1 immune response and memory generation

<p>Abstract</p> <p>Background</p> <p><it>Leishmania (Viannia) shawi </it>parasite was first characterized in 1989. Recently the protective effects of soluble leishmanial antigen (SLA) from <it>L. (V.) shawi </it>promastigotes were demonstrated using BALB/c mice, the susceptibility model for this parasite. In order to identify protective fractions, SLA was fractionated by reverse phase HPLC and five antigenic fractions were obtained.</p> <p>Methods</p> <p>F1 fraction was purified from L. (V.) shawi parasite extract by reverse phase HPLC. BALB/c mice were immunized once a week for two consecutive weeks by subcutaneous routes in the rump, using 25 μg of F1. After 1 and 16 weeks of last immunization, groups were challenged in the footpad with L. (V.) shawi promastigotes. After 2 months, those same mice were sacrificed and parasite burden, cellular and humoral immune responses were evaluated.</p> <p>Results</p> <p>The F1 fraction induced a high degree of protection associated with an increase in IFN-γ, a decrease in IL-4, increased cell proliferation and activation of CD8⁺T lymphocytes. Long-term protection was acquired in F1-immunized mice, associated with increased CD4⁺central memory T lymphocytes and activation of both CD4⁺and CD8⁺T cells. In addition, F1-immunized groups showed an increase in IgG2a levels.</p> <p>Conclusions</p> <p>The inductor capability of antigens to generate memory lymphocytes that can proliferate and secrete beneficial cytokines upon infection could be an important factor in the development of vaccine candidates against American Tegumentary Leishmaniasis.</p

TRH fails to antagonize the acute paralytic effects of intrathecal dynorphin A and substance P antagonists in the rat

Thyrotropin releasing hormone (TRH), which has been shown to improve neurologic recovery following cervical contusive spinal injury in cats, has also recently been reported to prevent the neuronal damage produced by the intrathecal (i.t.) administration of the substance P antagonist, spantide. Spantide and other substance P antagonists share with dynorphin A (DYN A)-related peptides the ability to produce an acute hindlimb paralysis after i.t. administration in the rat. By virtue of this effect, DYN A has been implicated in the secondary injury mechanisms that follow spinal trauma. Since TRH was shown to reduce the degree of histopathological injury caused by i.t. spantide, we investigated the ability of TRH to prevent or ameliorate the acute hindlimb paralysis produced by the i.t. injection of the substance P antagonists, (D-Arg1,D-Trp7,9,Leu11)-substance P (spantide) and (D-Arg1,D-Pro2,D-Trp7,9,Leu11)-substance P, and DYN A in rats. In this study, TRH failed to improve motor function or survival following i.t. injections of substance P antagonists or DYN A

Endogenous opioids in spinal cord injury: a critical evaluation

Based upon evidence that opioid antagonists improve neurological outcome following either traumatic or ischemic spinal cord injury, endogenous opioids have been implicated in the pathophysiology of these disorders. Naloxone improved both spinal cord perfusion and neurological function following traumatic spinal cord injury in cats, and was subsequently observed to improve neurological outcome following ischemic spinal cord injury in rabbits. Using several opioid antagonists with varied selectivities for different types of opioid receptors, it was suggested that kappa opioid receptors are involved in both these models of spinal cord injury. In addition, spinal cord trauma in rats is associated with increased concentrations of the endogenous kappa agonist dynorphin A, and increased kappa opioid receptor binding capacity localized to the injury site. Furthermore, dynorphin A induces hindlimb and tail flaccidity following intrathecal injection in rats. Thus, the pathophysiological effects of endogenous opioids in spinal cord injury have been proposed to involve dynorphin A interactions with kappa opioid receptors. However, disparities between the actions of intrathecally injected dynorphin A in rats and the presumed actions of endogenous dynorphin A in cat and rabbit spinal cord injury have been revealed in recent experiments. Paralysis resulting from intrathecal dynorphin A is not altered by opioid receptor antagonists or TRH, produced by non-opioid dynorphin A fragments but not by other selective kappa opioid agonists, and associated with non-opioid mediated reductions in spinal cord blood flow. Furthermore, despite reports of endogenous opioid changes following rat spinal cord trauma, in contrast to cats and rabbits, naloxone failed to improve neurological outcome following traumatic rat spinal cord injury. Thus, the specific endogenous opioids and opioid receptor types involved in spinal cord injury remain to be resolved, and do not appear to be universal among different models of spinal cord injury in different species. Additionally, dynorphin A may participate in spinal cord injury mechanisms in the rat through non-opioid actions