Protective efficacy of an IL-12-expressing baculoviral malaria vaccine.

Interleukin-12 (IL-12) plays an important role in antigen-specific adaptive immunity against Plasmodium sporozoites, and this requirement allows for a new approach to developing an effective malaria vaccine. In this study, we examined whether IL-12 could enhance protective efficacy of a baculovirus-based malaria vaccine. For this aim, a baculoviral vector expressing murine IL-12 (mIL-12) under the control of CMV promoter (BES-mIL-12-Spider) and a baculoviral vector expressing Plasmodium falciparum circumsporozoite protein (PfCSP) with post-transcriptional regulatory element of woodchuck hepatitis virus (BDES-sPfCSP2-WPRE-Spider) were generated. BES-mIL-12-Spider produced bioactive IL-12 which activates splenocytes, resulting in induction of IFN-γ. When co-immunized with BES-mIL-12-Spider and BDES-sPfCSP2-WPRE-Spider, the mouse number for high IgG2a/IgG1 ratios and the geometric mean in this group were both increased as compared with those of the other groups, indicating a shift towards a Th1-type response following immunization with BES-mIL-12-Spider. Finally, immunization with BDES-sPfCSP2-WPRE-Spider plus BES-mIL-12-Spider had a higher protective efficacy (73%) than immunization with BDES-sPfCSP2-WPRE-Spider alone (30%) against challenge with transgenic Plasmodium berghei sporozoites expressing PfCSP. These results suggest that co-administration of IL-12 expressing baculoviral vector, instead of IL-12 cDNA, with viral-vectored vaccines provides a new feasible vaccine platform to enhance Th1-type cellular immune responses against Plasmodium parasites.

The Plasmodium berghei sexual stage antigen PSOP12 induces anti-malarial transmission blocking immunity both in vivo and in vitro.

Anti-malarial transmission-blocking vaccines (TBVs) aim to inhibit the transmission of Plasmodium from humans to mosquitoes by targeting the sexual/ookinete stages of the parasite. Successful use of such interventions will subsequently result in reduced cases of malarial infection within a human population, leading to local elimination. There are currently only five lead TBV candidates under examination. There is a consequent need to identify novel antigens to allow the formulation of new potent TBVs. Here we describe the design and evaluation of a potential TBV (BDES-PbPSOP12) targeting Plasmodium berghei PSOP12 based on the baculovirus dual expression system (BDES), enabling expression of antigens on the surface of viral particles and within infected mammalian cells. In silico studies have previously suggested that PSOP12 (Putative Secreted Ookinete Protein 12) is expressed within the sexual stages of the parasite (gametocytes, gametes and ookinetes), and is a member of the previously characterized 6-Cys family of plasmodial proteins. We demonstrate that PSOP12 is expressed within the sexual/ookinete forms of the parasite, and that sera obtained from mice immunized with BDES-PbPSOP12 can recognize the surface of the male and female gametes, and the ookinete stages of the parasite. Immunization of mice with BDES-PbPSOP12 confers modest but significant transmission-blocking activity in vivo by active immunization (53.1% reduction in oocyst intensity, 10.9% reduction in oocyst prevalence). Further assessment of transmission-blocking potency ex vivo shows a dose-dependent response, with up to a 76.4% reduction in intensity and a 47.2% reduction in prevalence observed. Our data indicates that PSOP12 in Plasmodium spp. could be a potential new TBV target candidate, and that further experimentation to examine the protein within human malaria parasites would be logical.

A new member of the HCO3(-) transporter superfamily is an apical anion exchanger of beta-intercalated cells in the kidney.

The kidneys play pivotal roles in acid-base homeostasis, and the acid-secreting (alpha-type) and bicarbonate-secreting (beta-type) intercalated cells in the collecting ducts are major sites for the final modulation of urinary acid secretion. Since the H(+)-ATPase and anion exchanger activities in these two types of intercalated cells exhibit opposite polarities, it has been suggested that the alpha- and beta-intercalated cells are interchangeable via a cell polarity change. Immunohistological studies, however, have failed to confirm that the apical anion exchanger of beta-intercalated cells is the band 3 protein localized to the basolateral membrane of alpha-intercalated cells. In the present study, we show the evidence that a novel member of the anion exchanger and sodium bicarbonate cotransporter superfamily is an apical anion exchanger of beta-intercalated cells. Cloned cDNA from the beta-intercalated cells shows about 30% homology with anion exchanger types 1-3, and functional expression of this protein in COS-7 cells and Xenopus oocytes showed sodium-independent and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-insensitive anion exchanger activity. Furthermore, immunohistological studies revealed that this novel anion exchanger is present on the apical membrane of beta-intercalated cells, although some beta-intercalated cells were negative for AE4 staining. We conclude that our newly cloned transporter is an apical anion exchanger of the beta-intercalated cells, whereas our data do not exclude the possibility that there may be another form of anion exchanger in these cells.

Distal urinary acidification from Homer Smith to the present.

Since Smith's time, the essential role of collecting duct intercalated cells in controlling net acid excretion has been recognized. Rather than employing an H(+)-exchange mechanism, intercalated cells have V-ATPase on the plasma membrane and in plasmalemma-associated tubulovesicles, which functions in the bicarbonate reabsorption, regeneration, and bicarbonate secretion required for acid-base homeostasis. Several distinct mechanisms participate in regulating V-ATPase-driven H+ secretion in different cell types: (1) Renal epithelial cells have the capacity to express different structural forms of V-ATPase that have intrinsic differences in their enzymatic properties. 2) The kidney produces cytosolic regulatory proteins, capable of interacting directly with the V-ATPase, that may modify its activity. V-ATPases in different cell types may differ in the degree to which their activity is affected by regulatory factors, as a result of variations in V-ATPase structure. (3) In the alpha intercalated cell, the number of active V-ATPases on the luminal membrane is controlled in vivo by membrane vesicle-mediated traffic that may require unidentified mediators. In the beta intercalated cell, the number of active V-ATPases on the basolateral membrane may be controlled by regulated assembly and disassembly, responding directly to extracellular pH.

Physiology and biochemistry of the kidney vacuolar H+-ATPase.

Vacuolar H+-ATPases have an essential role in renal hydrogen ion secretion in the proximal tubule, collecting duct, and other segments of the nephron. Control of H+ transport is achieved by variations in the intrinsic properties of the renal H+-ATPases and by several cellular regulatory mechanisms, including redistribution of the enzyme both by vesicular traffic and regulated assembly and disassembly, and cytosolic regulatory proteins that interact directly with H+-ATPase. These mechanisms may provide a means for fine control of net acid excretion and for regulating vacuolar H+-ATPases residing on the plasma membrane independently from those in intracellular compartments.

Corticosterone increases Na(+)-K(+)-ATPase activity in rat cortical collecting ducts with inhibition of 11 beta-hydroxysteroid dehydrogenase.

To examine a physiological role of 11 beta-hydroxysteroid dehydrogenase (11OHSD) in the aldosterone target tissue, we measured Na(+)-K(+)-ATPase activity in the cortical collecting ducts (CCD) from adrenalectomized rats, which were treated with a physiological dose of corticosterone and/or carbenoxolone (an inhibitor of 11OHSD). The Na(+)-K(+)-ATPase activity in adrenalectomized rats was not significantly changed by either corticosterone alone or carbenoxolone alone, whereas its activity showed a significant increase only in the rats that received both corticosterone and carbenoxolone. In these rats, the plasma concentration of corticosterone was within the physiological range (10(-6) M) and the plasma carbenoxolone concentration was about 10(-7) M. Furthermore, the direct effect of carbenoxolone was examined in the microdissected CCD because it has been reported that carbenoxolone per se had an affinity for the aldosterone receptor. Na+K(+)-ATPase activity in the microdissected CCD was increased in a dose-dependent manner after a 3-hour incubation with carbenoxolone, and this effect was completely inhibited by canrenoic acid (an aldosterone antagonist). However, the minimal carbenoxolone concentration exerting a stimulatory effect was 10(-6) M, which is about 10 times higher than the plasma concentration of carbenoxolone in the in vivo treated rats. These results indicate that an inhibition of 11OHSD but not a direct action of carbenoxolone induces an increase in Na(+)-K(+)-ATPase activity, which is a well-known aldosterone effect in the CCD.

Effects of in vivo and in vitro alkali treatment on intracellular pH regulation of OMCDis cells.

To examine functional changes of the transporters in the inner stripe of the outer medullary collecting ducts (OMCDis) by the peritubular acid-base status, in vitro microperfusion using the acetoxymethyl ester of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein was performed. Cell alkalinization systems were assessed by the recovery rate (dpHi/dt) of intracellular pH (pHi) after intracellular acid loading by NH(4+)-NH3 prepulse with bath amiloride. In alkali-loaded rabbits (0.15 M NaHCO3 drinking for 14 days), dpHi/dt showed a significant decrease (1.80 +/- 0.29 pH units/s x 10(3)) compared with either control (3.30 +/- 0.59) or acid-loaded rabbits (0.15 M NH4Cl drinking for 14 days, 3.05 +/- 0.46). The difference of dpHi/dt between control and alkali-loaded rabbits was eliminated by lumen N-ethylmaleimide (NEM), suggesting that H+ pump activity was decreased. The effect of in vitro alkali treatment (50 mM HCO3-, pH 7.7) for 3-4 h was also examined. This incubation significantly decreased the dpHi/dt (1.83 +/- 0.35) compared with the time control experiments (3.18 +/- 0.28), whereas no significant difference was seen in the presence of lumen NEM. Anion exchanger activity, as determined from the pHi changes after Cl- addition to the bath, showed no significant change with in vivo or in vitro alkali treatment. The results indicate that cell function of the OMCDis is regulated in response to the peritubular acid-base environment via changes in the H(+)-adenosinetriphosphatase.

Isoproterenol stimulates Cl- current by a Gs protein-mediated process in beta-intercalated cells isolated from rabbit kidney.

To examine the effect of isoproterenol on Cl- current and its signal transduction pathway in beta-intercalated cells (beta-IC cell), peanut agglutinin (PNA) positive cells in culture were studied by the whole-cell clamp technique. We identified these cells as beta-IC cells by PNA-binding, cell alkalinization induced by Cl- free in the superfusate, and an increase in intracellular cAMP concentration by isoproterenol, but not by vasopressin. Application of isoproterenol in the voltage-clamp mode induced an activation of Cl- current in a dose-dependent fashion and its threshold concentration was in the order of 0.01 microM and ED50 was about 0.1 microM. This effect of isoproterenol was inhibited by atenolol, a beta-adrenergic blocker. Either extracellular application of forskolin or intracellular application of cAMP mimicked the action of isoproterenol. In the presence of forskolin or cAMP, isoproterenol caused little further activation of Cl- current. A synthetic inhibitor of protein kinase A (5-24 amide) inhibited the Cl- -channel activation by isoproterenol. Isoproterenol failed to activate the current in the presence of intracellular GDP beta S. By contrast, intracellular application of GTP gamma S rendered irreversible the Cl- -channel activation by brief exposure to isoproterenol. The present studies provide direct evidence that in the PNA-binding cell, probably the beta-IC cell, the stimulation of beta-adrenoceptor activates Cl- current through the signal transduction system involving G-protein, adenylate cyclase, cAMP, and protein kinase A.

Chronic DOC treatment enhances Na(+)-H+ exchanger activity of beta-intercalated cells in rabbit CCD.

Chronic deoxycorticosterone (DOC) treatment is known to increase HCO3- secretion in rabbit cortical collecting ducts (CCD). In this study, we examined whether changes in number or function of intercalated cells (ICC) are induced by DOC treatment. The number of total ICC [acetoxymethyl ester of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF/AM)-positive cells after its luminal loading], and beta-ICC (peanut agglutinin-positive cells) was not different between DOC and control groups in either initial CCD or terminal CCD. To evaluate the single-cell function of ICC, the rate of intracellular pH (pHi) recovery (dpHi/dt, pHU/s x 10(3)) after NH4+/NH3 prepulse was studied in the in vitro microperfused CCD in the presence of HCO3-/CO2 with BCECF/AM. The mean rate of dpHi/dt of beta-ICC in the DOC group was faster than that in the control group (6.19 +/- 0.36 vs. 4.30 +/- 0.41, P less than 0.005, respectively), whereas baseline pHi and buffer capacity were similar in the two groups. The inhibition of basolateral Na(+)-H+ exchanger with 1 mM amiloride eliminated the difference of dpHi/dt between the two groups, indicating the increased activity of basolateral Na(+)-H+ exchanger of beta-ICC in the DOC group. The correction of DOC-induced metabolic alkalosis by oral acid loading abolished the increase in Na+/H+ exchanger activity by chronic DOC treatment. These results suggest that DOC treatment induces a functional change in a single beta-ICC and that this functional change was induced by in vivo acid-base status.

Effect of isoproterenol on intracellular pH of the intercalated cells in the rabbit cortical collecting ducts.

To examine the mechanisms which regulate the functions of the intercalated cells (ICs) in the cortical collecting duct (CCD), the effect of isoproterenol on intracellular pH (pHi) of ICs was studied with the in vitro microperfused rabbit CCD, using the single cell pHi determination technique with fluorescent dye, 2',7'-bis-(2-carboxyethyl)-5(and-6)carboxyfluorescein. The pHi of beta-IC was significantly decreased with the addition of basolateral 10(-6) M isoproterenol (7.21 +/- 0.04 to 7.05 +/- 0.04), whereas alpha-IC did not show any change. This response of beta-IC to isoproterenol was dose-dependent and completely inhibited by the beta-blockers, atenolol or propranolol. The addition of forskolin or 8-Br-cAMP mimicked the effects of isoproterenol, suggesting that the activation of adenylate cyclase induced the decrease in pHi. The rate of pHi changes after the Cl- removal from the perfusate, which is considered to reflect the activity of luminal anion exchanger, was significantly higher with isoproterenol (0.032 +/- 0.009 pH unit/s) than that in the control (0.023 +/- 0.009 pH unit/s). The present studies provide direct evidence for the regulation of beta-IC function by beta-adrenergic receptor; and the luminal Cl-/HCO3- exchanger was considered to be stimulated by beta-agonist, directly.