Knockdown of the Sodium/Potassium ATPase Subunit Beta 2 Reduces Egg Production in the Dengue Vector, Aedes aegypti.

The Na+/K+ ATPase (NKA) is present in the cellular membrane of most eukaryotic cells. It utilizes energy released by ATP hydrolysis to pump sodium ions out of the cell and potassium ions into the cell, which establishes and controls ion gradients. Functional NKA pumps consist of three subunits, alpha, beta, and FXYD. The alpha subunit serves as the catalytic subunit while the beta and FXYD subunits regulate the proper folding and localization, and ion affinity of the alpha subunit, respectively. Here we demonstrate that knockdown of NKA beta subunit 2 mRNA (nkaß2) reduces fecundity in female Ae. aegypti. We determined the expression pattern of nkaß2 in several adult mosquito organs using qRT-PCR. We performed RNAi-mediated knockdown of nkaß2 and assayed for lethality, and effects on female fecundity. Tissue expression levels of nkaß2 mRNA were highest in the ovaries with the fat body, midgut and thorax having similar expression levels, while Malpighian tubules had significantly lower expression. Survival curves recorded post dsRNA injection showed a non-significant decrease in survival of nkaß2 dsRNA-injected mosquitoes compared to GFP dsRNA-injected mosquitoes. We observed a significant reduction in the number of eggs laid by nkaß2 dsRNA-injected mosquitoes compared to control mosquitoes. These results, coupled with the tissue expression profile of nkaß2, indicate that this subunit plays a role in normal female Ae. aegypti fecundity. Additional research needs to be conducted to determine the exact role played by NKAß2 in mosquito post-blood meal nutrient sensing, transport, yolk precursor protein (YPP) synthesis and yolk deposition.

General control nonderepressible 1 interacts with cationic amino acid transporter 1 and affects Aedes aegypti fecundity.

BACKGROUND: The amino acid transporter protein cationic amino acid transporter 1 (CAT1) is part of the nutrient sensor in the fat body of mosquitoes. A member of the SLC7 family of cationic amino acid transporters, it is paramount for the detection of elevated amino acid levels in the mosquito hemolymph after a blood meal and the subsequent changes in gene expression in the fat body. METHODS: We performed a re-annotation of Aedes aegypti cationic amino acid transporters (CATs) and selected the C-terminal tail of CAT1 to perform a yeast two-hybrid screen to identify putative interactors of this protein. One interesting interacting protein we identified was general control nonderepressible 1 (GCN1). We determined the expression pattern of GCN1 in several adult organs and structures using qRT-PCR and western blots. Finally, we knocked down GCN1 using double-stranded RNA and identified changes in downstream signaling intermediates and the effects of knockdown on vitellogenesis and fecundity. RESULTS: In a screen for Ae. aegypti CAT1-interacting proteins we identified GCN1 as a putative interactor. GCN1 is highly expressed in the ovaries and fat body of the mosquito. We provide evidence that eukaryotic translation initiation factor 2 subunit alpha (eIF2α) phosphorylation changed during vitellogenesis and that RNA interference knockdown of GCN1 in whole mosquitoes reduced egg clutch sizes of treated mosquitoes relative to controls. CONCLUSIONS: Aedes aegypti CAT1 and GCN1 are likely interacting partners and GCN1 is likely necessary for proper egg development. Our data suggest that GCN1 is part of a nutrient sensor mechanism in various mosquito tissues involved in vitellogenesis.

Exploratory phosphoproteomics profiling of Aedes aegypti Malpighian tubules during blood meal processing reveals dramatic transition in function.

Malpighian tubules, the renal organs of mosquitoes, facilitate the rapid dehydration of blood meals through aquaporin-mediated osmosis. We performed phosphoproteomics analysis of three Malpighian tubule protein-libraries (1000 tubules/sample) from unfed female mosquitoes as well as one and 24 hours after a blood meal. We identified 4663 putative phosphorylation sites in 1955 different proteins. Our exploratory dataset reveals blood meal-induced changes in phosphorylation patterns in many subunits of V-ATPase, proteins of the target of rapamycin signaling pathway, vesicle-mediated protein transport proteins, proteins involved in monocarboxylate transport, and aquaporins. Our phosphoproteomics data suggest the involvement of a variety of new pathways including nutrient-signaling, membrane protein shuttling, and paracellular water flow in the regulation of urine excretion. Our results support a model in which aquaporin channels translocate from intracellular vesicles to the cell membrane of stellate cells and the brush border membrane of principal cells upon blood feeding.

High efficiency ex vivo cloning of antigen-specific human effector T cells.

While cloned T cells are valuable tools for the exploration of immune responses against viruses and tumours, current cloning methods do not allow inferences to be made about the function and phenotype of a clone's in vivo precursor, nor can precise cloning efficiencies be calculated. Additionally, there is currently no general method for cloning antigen-specific effector T cells directly from peripheral blood mononuclear cells, without the need for prior expansion in vitro. Here we describe an efficient method for cloning effector T cells ex vivo. Functional T cells are detected using optimised interferon gamma capture following stimulation with viral or tumour cell-derived antigen. In combination with multiple phenotypic markers, single effector T cells are sorted using a flow cytometer directly into multi-well plates, and cloned using standard, non antigen-specific expansion methods. We provide examples of this novel technology to generate antigen-reactive clones from healthy donors using Epstein-Barr virus and cytomegalovirus as representative viral antigen sources, and from two melanoma patients using autologous melanoma cells. Cloning efficiency, clonality, and retention/loss of function are described. Ex vivo effector cell cloning provides a rapid and effective method of deriving antigen-specific T cells clones with traceable in vivo precursor function and phenotype.

Dendritic cell immunotherapy for stage IV melanoma.

Active boosting of the antitumour immune response of patients with solid malignancies has been tested in a large number of trials. Isolated complete clinical responses have been reported, however, they have not been replicated in subsequent studies. We recently reported objective clinical responses to a dendritic cell/irradiated autologous tumour cell 'vaccine' in patients with distant metastatic (stage IV) melanoma. Here we describe our experience in a second cohort of patients with stage IV melanoma, using this dendritic cell-based immunotherapy in a cryopreserved format. Of 46 patients enrolled into the study, three had complete remission of all detectable disease, and a further three had partial clinical responses. These data confirm that dendritic cell-based immunotherapy has potential as a therapy in a limited number of patients with stage IV melanoma. To our knowledge, this is the first demonstration that cryopreserved dendritic cells can elicit complete clinical responses in patients with advanced cancer. Our observations support randomized controlled trials to validate the findings.

Immunological characteristics correlating with clinical response to immunotherapy in patients with advanced metastatic melanoma.

Current treatment options for advanced metastatic melanoma are limited to experimental regimen that provide poor survival outcomes. Immunotherapy is a promising alternative and we recently reported a clinical trial in which 6 out of 19 patients enrolled had objective clinical responses to a fully autologous melanoma/dendritic cell vaccine. The mechanism of the vaccine is not well understood, but we hypothesized that general immunocompetence may be a determinant of clinical response. We therefore examined the immune status of an expanded series of 21 patients who displayed varying clinical responses to the melanoma/dendritic cell vaccine. Immunocompetence was assessed using in vitro assays of lymphocyte function: survival, proliferation and cytokine responses to mitogen stimulation as well as T-cell receptor zeta expression and lymphocyte subset analysis. Although lymphocytes from patients mostly performed comparably to age-matched and sex-matched controls, in some assays we identified significant differences between complete clinical responders and other patients, both before and following vaccination. Surprisingly, before vaccination, only lymphocytes from clinical responder patients showed impaired in vitro survival. Following vaccination, T lymphocyte survival improved and cells recovered their ability to produce the Th1-associated cytokines TNF and IFN-gamma in response to anti-CD3 stimulation in vitro. No increase in Th1 cytokine production was observed in lymphocytes from patients who experienced partial clinical responses or progressive disease. We conclude that, before vaccination, patients who go on to have complete responses have immune characteristics suggestive of high cell turnover and low Th1-associated cytokine production, and that these can be reversed with vaccination. These results have potential implications for future immunotherapeutic strategies.

Anatomical distribution of solar ultraviolet exposures among cyclists.

Exposure to solar ultraviolet (UV) radiation is the major environmental factor implicated in the development of melanoma and other skin cancers, as well as eye damage and skin photoaging. Outdoor recreational activities such as cycling are increasingly pursued for health benefits, however little information is available regarding potential adverse effects of excessive sun exposure in this setting, nor about the anatomical distribution of solar dose. Polysulphone badges (UV dosimeters) were attached to the head, backs of hands and ankles of 22 cyclists during a seven-day charity bicycle ride in Queensland, Australia. Average daily exposures exceeded one minimal erythemal dose (MED) at all body sites except the ankle. Significant differences in UV dose among the various body sites were noted, with highest exposures recorded on the top of the head. Mean doses received at the ankle (0.94 MED), back of the hand (1.28 MED) and side of the head (1.14 MED) were 51%, 71% and 63% of those received at the top of the head (1.80 MED), respectively. These data indicate that cycling exposes adherents to substantial doses of UV radiation. Moreover, our observations suggest that even vertically-oriented, potentially shaded sites such as the lower leg typically receive doses of solar radiation no less than half of maximally exposed sites.

(Pro)insulin-specific regulatory T cells.

Regulatory anti-diabetogenic T cells (T(reg)) can be induced by the mucosal administration of insulin or proinsulin peptides, in the non-obese diabetic (NOD) mouse model of autoimmune type 1 diabetes. Naso-respirtory insulin (which avoids insulin degradation) induces CD8+ alpha(alpha) TCR gamma(delta) T(reg) whereas peptides that bind to the NOD MHC class II molecule, I-Ag7, insulin B9-23 and proinsulin B24-C36, induce CD4+ T(regs) Following naso-respiratory delivery of insulin to NOD mice increased numbers of CD8+ gamma(delta) T cells expressing interleukin (IL)10 are detected in the pancreatic lymph nodes. Neonatal (3 day) thymectomy (NTX) dramatically accelerates diabetes development in NOD mice, associated with lymphopaenia and a block in the maturation of mucosal intrepithelial lymphocytes (IEL), especially extrathymic-derived CD8+ alpha(alpha) TCR gamma(delta) IEL. Regulatory anti-diabetogenic T cells cannot be elicited by naso-respiratory insulin in NTX-NOD mice. Reconstitution of NTX-NOD mice with CD8+ alpha(alpha) TCR gamma(delta) T cells prevents diabetes. CD8+ gamma(delta) T(reg) are conceivably physiological and insulin-specific, induced by exposure to insulin in maternal milk. These findings infer an immunoregulatory role for extrathymic-derived IEL, developing under the influence of the thymus and conditioned by early exposure to the exogenous environment.

Disabling an integral CTL epitope allows suppression of autoimmune diabetes by intranasal proinsulin peptide.

Insulin is a major target of the autoimmune response associated with destruction of pancreatic beta cells in type 1 diabetes. A peptide that spans the junction of the insulin B chain and the connecting (C) peptide in proinsulin has been reported to stimulate T cells from humans at risk for type 1 diabetes and autoimmune diabetes-prone NOD mice. Here we show that proinsulin B24-C36 peptide binds to I-A(g7), the MHC class II molecule of the NOD mouse, and, after intranasal administration, induces regulatory CD4(+) T cells that, in the absence of CD8(+) T cells, block the adoptive transfer of diabetes. Curiously, however, intranasal B24-C36 did not inhibit development of spontaneous diabetes in treated mice. We then determined that B24-C36, and its core sequence B25-C34, bind to K(d), the NOD mouse MHC class I molecule, and elicit CD8(+) CTLs. When the CD8(+) T lymphocyte epitope was truncated at the C34 valine anchor residue for binding to K(d), the residual CD4(+) T cell epitope, B24-C32/33, significantly inhibited diabetes development after a single intranasal dose. This study identifies a novel CTL epitope in proinsulin and demonstrates that the therapeutic potential of a "tolerogenic" autoantigen peptide can be compromised by the presence of an integral CTL epitope.

Transient blockade of CD40 ligand dissociates pathogenic from protective mucosal immunity.

Antigen administration via oral and other mucosal routes can suppress systemic immunity to the antigen and has been used to prevent experimental autoimmune disease. This approach may prove ineffective or even harmful if it leads to a concomitant induction of cytotoxic T lymphocytes (CTLs), and indeed, mucosal administration of the model antigen ovalbumin (OVA) has been shown to elicit CTL activation while simultaneously inducing oral tolerance. Here we show that induction by oral OVA of CTLs in wild-type mice, and of diabetes in mice expressing OVA transgenically in pancreatic beta cells, can be prevented by transiently blocking the CD40 ligand (CD40L). However, CD40L blockade did not diminish oral tolerance, as measured by suppression of systemic OVA-primed T cell proliferation, IFN-gamma secretion, and Ab production. Consistent with these findings, mice lacking CD40 expression could be orally tolerized to OVA. Transient CD40L blockade therefore dissociates pathogenic from protective immunity and should enhance the efficacy and safety of oral tolerance for preventing autoimmune disease.