Immunogenicity Evaluation of a Rationally Designed Polytope Construct Encoding HLA-A*0201 Restricted Epitopes Derived from Leishmania major Related Proteins in HLA-A2/DR1 Transgenic Mice: Steps toward Polytope Vaccine

Background

There are several reports demonstrating the role of CD8 T cells against Leishmania species. Therefore peptide vaccine might represent an effective approach to control the infection. We developed a rational polytope-DNA construct encoding immunogenic HLA-A2 restricted peptides and validated the processing and presentation of encoded epitopes in a preclinical mouse model humanized for the MHC-class-I and II.

Methods and Findings

HLA-A*0201 restricted epitopes from LPG-3, LmSTI-1, CPB and CPC along with H-2Kd restricted peptides, were lined-up together as a polytope string in a DNA construct. Polytope string was rationally designed by harnessing advantages of ubiquitin, spacers and HLA-DR restricted Th1 epitope. Endotoxin free pcDNA plasmid expressing the polytope was inoculated into humanized HLA-DRB1*0101/HLA-A*0201 transgenic mice intramuscularly 4 days after Cardiotoxin priming followed by 2 boosters at one week interval. Mice were sacrificed 10 days after the last booster, and splenocytes were subjected to ex-vivo and in-vitro evaluation of specific IFN-γ production and in-vitro cytotoxicity against individual peptides by ELISpot and standard chromium-51(⁵¹Cr) release assay respectively. 4 H-2Kd and 5 HLA-A*0201 restricted peptides were able to induce specific CD8 T cell responses in BALB/C and HLA-A2/DR1 mice respectively. IFN-γ and cytolytic activity together discriminated LPG-3-P1 as dominant, LmSTI-1-P3 and LmSTI-1-P6 as subdominant with both cytolytic activity and IFN-γ production, LmSTI-1-P4 and LPG-3-P5 as subdominant with only IFN-γ production potential.

Conclusions

Here we described a new DNA-polytope construct for Leishmania vaccination encompassing immunogenic HLA-A2 restricted peptides. Immunogenicity evaluation in HLA-transgenic model confirmed CD8 T cell induction with expected affinities and avidities showing almost efficient processing and presentation of the peptides in relevant preclinical model. Further evaluation will determine the efficacy of this polytope construct protecting against infectious challenge of Leishmania. Fortunately HLA transgenic mice are promising preclinical models helping to speed up immunogenicity analysis in a human related mouse model.     

Rad21l1 cohesin subunit is dispensable for spermatogenesis but not oogenesis in zebrafish

During meiosis I, ring-shaped cohesin complexes play important roles in aiding the proper segregation of homologous chromosomes. RAD21L is a meiosis-specific vertebrate cohesin that is required for spermatogenesis in mice but is dispensable for oogenesis in young animals. The role of this cohesin in other vertebrate models has not been explored. Here, we tested if the zebrafish homolog Rad21l1 is required for meiotic chromosome dynamics during spermatogenesis and oogenesis. We found that Rad21l1 localizes to unsynapsed chromosome axes. It is also found between the axes of the mature tripartite synaptonemal complex (SC) in both sexes. We knocked out rad21l1 and found that nearly all rad21l1^-/- mutants develop as fertile males, suggesting that the mutation causes a defect in juvenile oogenesis, since insufficient oocyte production triggers female to male sex reversal in zebrafish. Sex reversal was partially suppressed by mutation of the checkpoint gene tp53, suggesting that the rad21l1 mutation activates Tp53-mediated apoptosis or arrest in females. This response, however, is not linked to a defect in repairing Spo11-induced double-strand breaks since deletion of spo11 does not suppress the sex reversal phenotype. Compared to tp53 single mutant controls, rad21l1^-/- tp53^-/- double mutant females produce poor quality eggs that often die or develop into malformed embryos. Overall, these results indicate that the absence of rad21l1^-/- females is due to a checkpoint-mediated response and highlight a role for a meiotic-specific cohesin subunit in oogenesis but not spermatogenesis.     

Down-Regulation of the Epithelial Na+ Channel ENaC by Janus kinase 2

Janus kinase-2 (JAK2), a signaling molecule mediating effects of various hormones including leptin and growth hormone, has previously been shown to modify the activity of several channels and carriers. Leptin is known to inhibit and growth hormone to stimulate epithelial Na⁺ transport, effects at least partially involving regulation of the epithelial Na⁺ channel ENaC. However, no published evidence is available regarding an influence of JAK2 on the activity of the epithelial Na⁺ channel ENaC. In order to test whether JAK2 participates in the regulation of ENaC, cRNA encoding ENaC was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild type JAK2, gain-of-function ^V617FJAK2 or inactive ^K882EJAK2. Moreover, ENaC was expressed with or without the ENaC regulating ubiquitin ligase Nedd4-2 with or without JAK2, ^V617FJAK2 or ^K882EJAK2. ENaC was determined from amiloride (50 μM)-sensitive current (I _amil) in dual electrode voltage clamp. Moreover, I _amil was determined in colonic tissue utilizing Ussing chambers. As a result, the I _amil in ENaC-expressing oocytes was significantly decreased following coexpression of JAK2 or ^V617FJAK2, but not by coexpression of ^K882EJAK2. Coexpression of JAK2 and Nedd4-2 decreased I _amil in ENaC-expressing oocytes to a larger extent than coexpression of Nedd4-2 alone. Exposure of ENaC- and JAK2-expressing oocytes to JAK2 inhibitor AG490 (40 μM) significantly increased I _amil. In colonic epithelium, I _amil was significantly enhanced by AG490 pretreatment (40 μM, 1 h). In conclusion, JAK2 is a powerful inhibitor of ENaC.     

Effects of manipulation of water and nitrogen regime on the water relations of the desert shrub Larrea tridentata

Summary Water and nitrogen regimes of Larrea tridentata shrubs growing in the field were manipulated during an annual cycle. Patterns of leaf water status, leaf water relations characteristics, and stomatal behavior were followed concurrently. Large variations in leaf water status in both irrigated and nonirrigated individuals were observed. Predawn and midday leaf water potentials of nonirrigated shrubs were lowest except when measurements had been preceded by significant rainfall. Despite the large seasonal variation in leaf water status, reasonably constant, high levels of turgor were maintained. Pressure-volume curve analysis suggested that changes in the bulk leaf osmotic potential at full turgor were small and that nearly all of the turgor adjustment was due to tissue elastic adjustment. The increase in tissue elasticity with increasing water deficit manifested itself as a decrease in the relative water content at zero turgor and as a decrease in the tissue bulk elastic modulus. Because of large hydration-induced displacement in the osmotic potential and relative water content at zero turgor, it was necessary to use shoots in their natural state of hydration for pressure-volume curve determinations. Large diurnal and seasonal differences in maximum stomatal conductance were observed, but could not easily be attributed to variations in leaf water potential or leaf water relations characteristics such as the turgor loss point. The single factor which seemed to account for most of the diurnal and seasonal differences in maximum stomatal conductance between individual shrubs was an index of soil/root/ shoot hydraulic resistance. Daily maximum stomatal conductance was found to decrease with increasing soil/root/ shoot hydraulic resistance. This pattern was most consistent if the hydraulic resistance calculation was based on an estimate of total canopy transpiration rather than the more commonly used transpiration per unit leaf area. The reasons for this are discussed. It is suggested that while stomatal aperture necessarily represents a major physical resistance controlling transpiration, plant hydraulic resistance may represent the functional resistance through its effects on stomatal aperture.     

Analysis of mitochondrial DNA from somatic hybrid cell lines of Saccharum officinarum (sugarcane) and Pennisetum americanum (pearl millet)

Mitochondrial DNA (mtDNA) from cell suspension cultures of two intergeneric somatic hybrids of Pennisetum americanum (pearl millet) + Saccharum officinarum (sugarcane) was examined by restriction endonuclease digestion and hybridization with sorghum mtDNA cosmids. The mtDNA of one somatic hybrid was indistinguishable from that of pearl millet, while the second exhibited a combination of parental mtDNAs, suggesting mitochondrial fusion. Several novel, possibly recombinant, mtDNA restriction fragments were detected in this hybrid, which may have resulted from intergenmic recombination.Florida Agriculture Experiment Station Journal Series No: 8090.     

Splenocytes Seed Bone Marrow of Myeloablated Mice: Implication for Atherosclerosis

Extramedullary hematopoiesis has been shown to contribute to the pathogenesis of a variety of diseases including cardiovascular diseases. In this process, the spleen is seeded with mobilized bone marrow cells that augment its hematopoietic ability. It is unclear whether these immigrant cells that are produced/reprogrammed in spleen are similar or different from those found in the bone marrow. To begin to understand this, we investigated the relative potency of adult splenocytes per se to repopulate bone marrow of lethally-irradiated mice and its functional consequences in atherosclerosis. The splenocytes were harvested from GFP donor mice and transplanted into myeloablated wild type recipient mice without the inclusion of any bone marrow helper cells. We found that adult splenocytes repopulated bone marrow of myeloablated mice and the transplanted cells differentiated into a full repertoire of myeloid cell lineages. The level of monocytes/macrophages in the bone marrow of recipient mice was dependent on the cell origin, i.e., the donor splenocytes gave rise to significantly more monocytes/macrophages than the donor bone marrow cells. This occurred despite a significantly lower number of hematopoietic stem cells being present in the donor splenocytes when compared with donor bone marrow cells. Atherosclerosis studies revealed that donor splenocytes displayed a similar level of atherogenic and atheroprotective activities to those of donor bone marrow cells. Cell culture studies showed that the phenotype of macrophages derived from spleen is different from those of bone marrow. Together, these results demonstrate that splenocytes can seed bone marrow of myeloablated mice and modulate atherosclerosis. In addition, our study shows the potential of splenocytes for therapeutic interventions in inflammatory disease.     

Diets of giants: the nutritional value of sauropod diet during the Mesozoic

A major uncertainty in estimating energy budgets and population densities of extinct animals is the carrying capacity of their ecosystems, constrained by net primary productivity (NPP) and its digestible energy content. The hypothesis that increases in NPP due to elevated atmospheric CO₂ contributed to the unparalleled size of the sauropods has recently been rejected, based on modern studies on herbivorous insects that imply a general, negative correlation of diet quality and increasing CO₂. However, the nutritional value of plants grown under elevated CO₂ levels might be very different for vertebrate megaherbivores than for insects. Here we show plant species‐specific responses in metabolizable energy and nitrogen content, equivalent to a two‐fold variation in daily food intake estimates for a typical sauropod, for dinosaur food plant analogues grown under CO₂ concentrations spanning estimates for Mesozoic atmospheric concentrations. Our results potentially rebut the hypothesis that constraints on sauropod diet quality were driven by Mesozoic CO₂ concentration.