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Intracellular pH (pHi) regulation in the brain is important in both physiological and physiopathological conditions because changes in pHi generally result in altered neuronal excitability. In this review, we will cover 4 major areas: (1) The effect of pHi on cellular processes in the brain, including channel activity and neuronal excitability. (2) pHi homeostasis and how it is determined by the balance between rates of acid loading (J L) and extrusion (J E). The balance between J E and J L determine steady-state pHi, as well as the ability of the cell to defend pHi in the face of extracellular acid-base disturbances (e.g., metabolic acidosis). (3) The properties and importance of members of the SLC4 and SLC9 families of acid-base transporters expressed in the brain that contribute to J L (namely the Cl-HCO3 exchanger AE3) and J E (the Na-H exchangers NHE1, NHE3, and NHE5 as well as the Na(+)- coupled HCO3 (-) transporters NBCe1, NBCn1, NDCBE, and NBCn2). (4) The effect of acid-base disturbances on neuronal function and the roles of acid-base transporters in defending neuronal pHi under physiopathologic conditions.

Mammalian glycosylated rhesus (Rh) proteins include the erythroid RhAG and the nonerythroid RhBG and RhCG. RhBG and RhCG are expressed in multiple tissues, including hepatocytes and the collecting duct (CD) of the kidney. Here, we expressed human RhAG, RhBG and RhCG in Xenopus oocytes (vs. H2O-injected control oocytes) and used microelectrodes to monitor the maximum transient change in surface pH (DpHS) caused by exposing the same oocyte to 5 % CO₂/33 mM HCO₃⁻ (an increase) or 0.5 mM NH₃/NH₄⁺ (a decrease). Subtracting the respective values for day-matched, H₂O-injected control oocytes yielded channel-specific values (*). (ΔpH*(S))(CO₂) and (-ΔpH*(S))(NH₃) were each significantly >0 for all channels, indicating that RhBG and RhCG--like RhAG--can carry CO₂ and NH₃. We also investigated the role of a conserved aspartate residue, which was reported to inhibit NH₃ transport. However, surface biotinylation experiments indicate the mutants RhBG(D178N) and RhCG(D177N) have at most a very low abundance in the oocyte plasma membrane. We demonstrate for the first time that RhBG and RhCG--like RhAG--have significant CO₂ permeability, and we confirm that RhAG, RhBG and RhCG all have significant NH₃ permeability. However, as evidenced by (ΔpH*(S))(CO₂)/ (-ΔpH*(S))(NH₃) values, we could not distinguish among the CO₂/ NH₃ permeability ratios for RhAG, RhBG and RhCG. Finally, we propose a mechanism whereby RhBG and RhCG contribute to acid secretion in the CD by enhancing the transport of not only NH₃ but also CO₂ across the membranes of CD cells.

ERβ1 is often down-regulated in breast cancer compared to normal breast but mechanisms surrounding this are unclear. We examined whether loss of heterozygosity (LOH) or methylation at ERβ promoters (0N, 0K) and/or untranslated exon 0N were involved in ERβ down-regulation in breast cancer tissues and cell lines and if treatment with the de-methylating agent 5-aza-deoxycytidine and/or the histone deacetylase inhibitor Trichostatin A could influence expression in vitro. We found no evidence of correlation between LOH at 14q22-24 (genomic locus containing ERβ/ESR2), and ERβ1 expression in primary breast cancers. A negative correlation between ERβ1 mRNA expression and methylation status was observed for promoter 0N in BT-20, MDA-MB-453 and T47D cells. Promoter 0K was consistently unmethylated. In primary breast tumours, methylation of the untranslated exon 0N, downstream of promoter 0N, but not of promoter 0N itself, correlated with down-regulation of ERβ. In MDA-MB-453 cells, treatment with 5-aza-deoxycytidine was sufficient to induce ERβ1 expression from the 0N promoter while in BT-20 both agents were required. Examination of various sites on ESR2 highlighted epigenetic but not genetic regulation of ERβ1. In particular methylation adjacent to promoter 0N was a key regulatory event for ERβ1 silencing. A combination of de-methylating agents and histone deacetylase inhibitors fully restored ERβ1 expression which may offer a novel therapeutic angle for breast cancer management.

The SLC4A7 gene encodes the electroneutral sodium/HCO3 cotransporter NBCn1, which plays important physiological and pathophysiological roles in many cell types. Previous work identified six NBCn1 variants differing in the sequence of the extreme N terminus--MEAD in rat only, MERF in human only--as well as in the optional inclusion of cassettes I, II, and III. Earlier work also left open the question of whether optional structural elements (OSEs) affect surface abundance or intrinsic (per-molecule) transport activity. Here, we demonstrate for the first time that SLC4A7 from one species can express both MEAD- and MERF-NBCn1. We also identify a novel cassette IV of 20 aa, and extend by 10 the number of full-length NBCn1 variants. The alternative N termini and four cassettes could theoretically produce 32 major variants. Moreover, we identify a group of cDNAs predicted to encode just the cytosolic N-terminal domain (Nt) of NBCn1. A combination of electrophysiology and biotinylation shows that the OSEs can affect surface abundance and intrinsic HCO3(-) transport activity of NBCn1, as expressed in Xenopus oocytes. Specifically, MEAD tends to increase whereas novel cassette IV reduces surface abundance. Cassettes II, III and novel cassette IV all appear to increase the intrinsic activity of NBCn1.

The characterization of skin mechanics has many clinical implications and has been an active area of research for the past few decades. Biomechanical models have evolved from earlier empirical models to state-of-the-art structural models that provide linkage between tissue microstructure and macroscopic stress-strain response. To maximize the accuracy and predictive capabilities of such computational models, there is a need to reliably identify often a large number of unknown model parameters. This is critically dependent on the availability of experimental data that cover an extensive range of different deformation modes, and quantification of internal structural features, such as collagen orientation. To this end, future challenges should include the ongoing development of noninvasive instrumentation and imaging modalities for in vivo skin measurements. We highlight the important concept of tightly integrating computational models, instrumentation, and imaging modalities into a single platform to investigate skin biomechanics., (Copyright © 2013 Wiley Periodicals, Inc.)

In the basolateral membrane of proximal-tubule cells, NBCe1-A (SLC4A4, variant A), operating with an apparent Na(+):HCO(3)(-) stoichiometry of 1:3, contributes to the reclamation of HCO(3)(-) from the glomerular filtrate, thereby preventing whole body acidosis. Others have reported that NBCe1-like activity in human, rabbit, and rat renal preparations is substantially influenced by lithium, sulfite, oxalate, and harmaline. These data were taken as evidence for the presence of distinct Na(+) and CO(3)(2-) binding sites in NBCe1-A, favoring a model of 1 Na(+):1 HCO(3)(-):1 CO(3)(2-). Here, we reexamine these findings by expressing human or rabbit NBCe1-A clones in Xenopus oocytes. In oocytes, NBCe1-A exhibits a 1:2 stoichiometry and could operate in one of five thermodynamically equivalent transport modes: 1) cotransport of Na(+) + 2 HCO(3)(-), 2) cotransport of Na(+) + CO(3)(2-), 3) transport of NaCO(3)(-), 4) exchange of Na(+) + HCO(3)(-) for H(+), or 5) HCO(3)(-)-activated exchange of Na(+) for 2 H(+). In contrast to the behavior of NBCe1-like activity in renal preparations, we find that cloned NBCe1-A is only slightly stimulated by Li(+), not at all influenced by sulfite or oxalate, and only weakly inhibited by harmaline. These negative data do not uniquely support any of the five models above. In addition, we find that NBCe1-A mediates a small amount of Na(+)-independent NO(3)(-) transport and that NBCe1-A is somewhat inhibited by extracellular benzamil. We suggest that the features of NBCe1-like activity in renal preparations are influenced by yet-to-be-identified renal factors. Thus the actual ionic substrates of NBCe1 remain to be identified.

The mammalian Slc4 (Solute carrier 4) family of transporters is a functionally diverse group of 10 multi-spanning membrane proteins that includes three Cl-HCO3 exchangers (AE1-3), five Na(+)-coupled HCO3(-) transporters (NCBTs), and two other unusual members (AE4, BTR1). In this review, we mainly focus on the five mammalian NCBTs-NBCe1, NBCe2, NBCn1, NDCBE, and NBCn2. Each plays a specialized role in maintaining intracellular pH and, by contributing to the movement of HCO3(-) across epithelia, in maintaining whole-body pH and otherwise contributing to epithelial transport. Disruptions involving NCBT genes are linked to blindness, deafness, proximal renal tubular acidosis, mental retardation, and epilepsy. We also review AE1-3, AE4, and BTR1, addressing their relevance to the study of NCBTs. This review draws together recent advances in our understanding of the phylogenetic origins and physiological relevance of NCBTs and their progenitors. Underlying these advances is progress in such diverse disciplines as physiology, molecular biology, genetics, immunocytochemistry, proteomics, and structural biology. This review highlights the key similarities and differences between individual NCBTs and the genes that encode them and also clarifies the sometimes confusing NCBT nomenclature.

The SLC4 family consists of 10 genes (SLC4A1-5; SLC4A7-11). All encode integral membrane proteins with very similar hydropathy plots-consistent with 10-14 transmembrane segments. Nine SLC4 members encode proteins that transport HCO3(-) (or a related species, such as CO3(2-)) across the plasma membrane. Functionally, eight of these proteins fall into two major groups: three Cl-HCO3 exchangers (AE1-3) and five Na(+)-coupled HCO3(-) transporters (NBCe1, NBCe2, NBCn1, NBCn2, NDCBE). Two of the Na(+)-coupled transporters (NBCe1, NBCe2) are electrogenic; the other three Na(+)-coupled HCO3(-) transporters and all three AEs are electroneutral. In addition, two other SLC4 members (AE4, SLC4A9 and BTR1, SLC4A11) do not yet have a firmly established function. Most, though not all, SLC4 members are functionally inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS). SLC4 proteins play important roles many modes of acid-base homeostasis: the carriage of CO2 by erythrocytes, the transport of H(+) or HCO3(-) by several epithelia, as well as the regulation of cell volume and intracellular pH., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Activation of Na(+),HCO3(-) cotransport in vascular smooth muscle cells (VSMCs) contributes to intracellular pH (pH(i)) control during artery contraction, but the signaling pathways involved have been unknown. We investigated whether physical and functional interactions between the Na(+),HCO3(-) cotransporter NBCn1 (slc4a7) and the Ca(2+)/calmodulin-activated serine/threonine phosphatase calcineurin exist and play a role for pHi control in VSMCs. Using a yeast two-hybrid screen, we found that splice cassette II from the N terminus of NBCn1 interacts with calcineurin Aβ. When cassette II was truncated or mutated to disrupt the putative calcineurin binding motif PTVVIH, the interaction was abolished. Native NBCn1 and calcineurin Aβ co-immunoprecipitated from A7r5 rat VSMCs. A peptide (acetyl-DDIPTVVIH-amide), which mimics the putative calcineurin binding motif, inhibited the co-immunoprecipitation whereas a mutated peptide (acetyl-DDIATAVAA-amide) did not. Na(+),HCO3(-) cotransport activity was investigated in VSMCs of mesenteric arteries after an NH4(+) prepulse. During depolarization with 50 mM extracellular K(+) to raise intracellular [Ca(2+)], Na(+),HCO3(-) cotransport activity was inhibited 20-30% by calcineurin inhibitors (FK506 and cyclosporine A). FK506 did not affect Na(+),HCO3(-) cotransport activity in VSMCs when cytosolic [Ca(2+)] was lowered by buffering, nor did it disrupt binding between NBCn1 and calcineurin Aβ. FK506 augmented the intracellular acidification of VSMCs during norepinephrine-induced artery contractions. No physical or functional interactions between calcineurin Aβ and the Na(+)/H(+) exchanger NHE1 were observed in VSMCs. In conclusion, we demonstrate a physical interaction between calcineurin Aβ and cassette II of NBCn1. Intracellular Ca(2+) activates Na(+),HCO3(-) cotransport activity in VSMCs in a calcineurin-dependent manner which is important for protection against intracellular acidification.

Determining the effective concentration (i.e., activity) of ions in and around living cells is important to our understanding of the contribution of those ions to cellular function. Moreover, monitoring changes in ion activities in and around cells is informative about the actions of the transporters and/or channels operating in the cell membrane. The activity of an ion can be measured using a glass microelectrode that includes in its tip a liquid-membrane doped with an ion-selective ionophore. Because these electrodes can be fabricated with tip diameters that are less than 1 μm, they can be used to impale single cells in order to monitor the activities of intracellular ions. This review summarizes the history, theory, and practice of ion-selective microelectrode use and brings together a number of classic and recent examples of their usefulness in the realm of physiological study.

The renal electrogenic Na(+)/HCO(3)(−) cotransporter (NBCe1-A) contributes to the basolateral step of transepithelial HCO(3)(−) reabsorption in proximal tubule epithelia, contributing to the buffering of blood pH. Elsewhere in the body (e.g. muscle cells) NBCe1 variants contribute to, amongst other processes, maintenance of intracellular pH. Others have described a homozygous mutation in NBCe1 (NBCe1-A p.Ala799Val) in an individual with severe proximal renal tubular acidosis (pRTA; usually associated with defective HCO(3)(−) reabsorption in proximal tubule cells) and hypokalaemic periodic paralysis (hypoPP; usually associated with leaky cation channels in muscle cells). Using biotinylation and two-electrode voltage-clamp on Xenopus oocytes expressing NBCe1, we demonstrate that the mutant NBCe1-A (A(A799V)) exhibits a per-molecule transport defect that probably contributes towards the observed pRTA. Furthermore, we find that A(A799V) expression is associated with an unusual HCO(3)(−)-independent conductance that, if associated with mutant NBCe1 in muscle cells, could contribute towards the appearance of hypokalaemic paralysis in the affected individual. We also study three novel lab mutants of NBCe1-A: p.Ala799Ile, p.Ala799Gly and p.Ala799Ser. All three exhibit a per-molecule transport defect, but only A(A799I) exhibits an A(A799V)-like ion conductance. A(A799G) and A(A799S) exhibit unusual outward rectification in their HCO(3)(−)-dependent conductance and A(A799G) exhibits reduced sensitivity to both DIDS and tenidap. A799G is the first mutation shown to affect the apparent tenidap affinity of NBCe1. Finally we show that A(A799V) and A(A799I), which accumulate poorly in the plasma membrane of oocytes, exhibit signs of abnormal intracellular accumulation in a non-polarized renal cell-line.

Two maneuvers known to stimulate electrogenic sodium bicarbonate cotransporter 1 (NBCe1) activity are 1) deletion from the cytosolic amino-terminus (Nt) of NBCe1-C of an 87-amino acid sequence that contains an autoinhibitory domain (AID); and 2) binding of the protein IRBIT to elements within the same 87-amino acid module in a different variant, NBCe1-B. Helpful to understanding the relationship between these two phenomena would be an appreciation of the relative magnitude of stimulation caused by each maneuver for the same NBCe1 variant. In the present study, we performed two-electrode voltage-clamp on Xenopus oocytes expressing human NBCe1-B constructs, with and without human IRBIT constructs. We find that removal of the AID stimulates NBCe1-B to the same extent as coexpression of wild-type IRBIT. The potency of wild-type IRBIT apparently is reduced by the action of endogenous oocyte protein phosphatases: a mutant IRBIT that cannot be influenced by the action of protein phosphatase-1 stimulates NBCe1-B to an extent 50% greater than can be achieved by removal of the NBCe1-B AID. Thus the stimulatory effect of IRBIT cannot be explained solely by masking of autoinhibitory determinants within the AID. Finally, we find that an NBCe1-B construct that lacks amino acid residues 2-16 of the Nt is fully autoinhibited, but cannot be stimulated by IRBIT, indicating that autoinhibitory and IRBIT-binding determinants within the cytosolic Nt are not identical.

Identifying the mechanical properties of the skin has been the subject of much study in recent years, as such knowledge can provide insight into wound healing, wrinkling and minimization of scarring through surgical planning.

The recent publication of the apo-, closed-state 3D crystal structure of zebrafish (zf) P2X4.1 has not only revolutionized the P2X research field, but also highlighted the need for further crystal structures, of receptors in different activation states, so that we can gain a complete molecular understanding of ion channel function. zfP2X4.1 was selected as a 3D-crystallization candidate because of its ability to form stable trimers in detergent solution, and purified from over-expression in baculovirus-infected Spodoptera frugiperda (Sf9) insect cells. In this work, we have used a similar approach to express both human P2X4 (hP2X4) and Dictyostelium discoideum P2XA (DdP2XA) in Sf9 cells. Although hP2X4 did not form stable trimers in detergent solution, both receptors bound to ATP-coupled resins, indicating that their extracellular domains were folded correctly. DdP2XA formed strong trimers in detergent solution, and we were able to selectively purify trimers using preparative electrophoresis, and build a 21Å-resolution 3D structure using transmission electron microscopy and single particle analysis. Although the structure of DdP2XA possessed similar dimensions to those of the previously determined low-resolution hP2X4 structure and the zfP2X4.1 crystal structure, N-glycosylation mutagenesis and molecular modeling indicated differences between N-glycan usage and predicted accessibility in models of DdP2XA based on the zfP2X4.1 crystal structure. Our data demonstrate that DdP2XA expressed in insect cells retains ATP-binding capacity after detergent solubilization, is an ideal candidate for structural study, and possesses a significantly different 3D structure to that of both hP2X4 and zfP2X4.1., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Purpose: While secretagogue-induced diarrhea is rich in chloride (Cl(-)) and bicarbonate (HCO(3) (-)) anions, little is known about diarrhea or its anionic composition following irradiation. We performed studies to characterize the differences between cyclic adenosine monophosphate (cAMP)-stimulated anion secretions in irradiated and non-irradiated mice., Materials and Methods: HCO(3) (-) secretion was examined in basal, cAMP-stimulated, and irradiated jejunal tissues from BALB/c (Bagg albino) mice. The abdomens of the mice were γ-irradiated using a caesium-137 source., Results: Ussing-chamber experiments performed in an HCO(3)(-)-containing, Cl(-)-free solution on the bath side showed inhibition of HCO(3)(-) in irradiated mice. Non-irradiated mice exhibited bumetanide-sensitive and insensitive current, while irradiated mice displayed bumetanide-sensitive current. pH-stat experiments showed inhibition of basal and cAMP-stimulated HCO(3)(-) secretions in irradiated mice. Immunohistochemistry and Western blot analysis displayed a sodium-bicarbonate cotransporter expression in the villus and not the crypt of non-irradiated mice, while its expression and protein levels decreased in irradiated mice., Conclusions: Anion secretions in irradiated mice, being primarily Cl(-) and minimally HCO(3)(-), differ from that of secretagogue-induced anion secretions. Understanding anion loss will help us correct electrolyte imbalances, while reduced HCO(3)(-) secretion in the upper-gastrointestinal tract might also have implications for irradiation-induced nausea and vomiting.

We have previously shown that a hydrophobic alkylating compound, 1,5-iodonaphthyl-azide (INA) can efficiently inactivate the virulent strain of Venezuelan equine encephalitis virus (VEEV), V3000 in vitro. In this study, we have evaluated the safety of INA-inactivated V3000 and V3526 and the protective efficacy of INA-inactivated V3000. INA-inactivated V3000 and V3526 did not cause disease in suckling mice. RNA isolated from the INA-inactivated V3000 and V3526 was also not infectious. Immunization of adult mice with INA-inactivated V3000 induced an anti-VEEV antibody response and protected mice from virulent VEEV challenge. The protective efficacy of INA-inactivated V3000 increased with the use of adjuvants. Results suggest that inactivation of enveloped viruses by INA may occur by two independent mechanisms and the INA-inactivated VEEV elicit a protective antibody response in mice., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

We have constructed a thermal stereoscope utilizing three digital SLR cameras and an infrared camera for rapid surface reconstruction of diabetic foot geometry and temperature distribution. A structured light pattern is projected on to the foot to provide approximately 2500 reconstructed points. The reconstructed point cloud is then fitted to a finite element model, producing root mean squared errors of less than 0.4 mm.

Six monoclonal antibodies were isolated that exhibited specificity for a furin cleavage site deletion mutant (V3526) of Venezuelan equine encephalitis virus (VEEV). These antibodies comprise a single competition group and bound the E3 glycoprotein of VEEV subtype I viruses but failed to bind the E3 glycoprotein of other alphaviruses. These antibodies neutralized V3526 virus infectivity but did not neutralize the parental strain of Trinidad donkey (TrD) VEEV. However, the E3-specific antibodies did inhibit the production of virus from VEEV TrD-infected cells. In addition, passive immunization of mice demonstrated that antibody to the E3 glycoprotein provided protection against lethal VEEV TrD challenge. This is the first recognition of a protective epitope in the E3 glycoprotein. Furthermore, these results indicate that E3 plays a critical role late in the morphogenesis of progeny virus after E3 appears on the surfaces of infected cells.

The central role of the multifunctional protein nephrin within the macromolecular complex forming the glomerular slit diaphragm is well established, but the mechanisms linking the slit diaphragm to the cytoskeleton and to the signaling pathways involved in maintaining the integrity of the glomerular filter remain incompletely understood. Here, we report that nephrin interacts with the bicarbonate/chloride transporter kidney anion exchanger 1 (kAE1), detected by yeast two-hybrid assay and confirmed by immunoprecipitation and co-localization studies. We confirmed low-level glomerular expression of kAE1 in human and mouse kidneys by immunoblotting and immunofluorescence microscopy. We observed less kAE1 in human glomeruli homozygous for the NPHS1(FinMaj) nephrin mutation, whereas kAE1 expression remained unchanged in the collecting duct. We could not detect endogenous kAE1 expression in NPHS1(FinMaj) podocytes in primary culture, but heterologous re-introduction of wild-type nephrin into these podocytes rescued kAE1 expression. In kidneys of Ae1(-/-) mice, nephrin abundance was normal but its distribution was altered along with the reported kAE1-binding protein integrin-linked kinase (ILK). Ae1(-/-) mice had increased albuminuria with glomerular enlargement, mesangial expansion, mesangiosclerosis, and expansion of the glomerular basement membrane. Glomeruli with ILK-deficient podocytes also demonstrated altered AE1 and nephrin expression, further supporting the functional interdependence of these proteins. These data suggest that the podocyte protein kAE1 interacts with nephrin and ILK to maintain the structure and function of the glomerular basement membrane.

The five known Na-coupled HCO(3)(-) transporters (NCBTs) of the solute carrier 4 (SLC4) family play important roles in pH regulation and transepithelial HCO(3)(-) transport. Nearly all of the NCBTs have multiple splice variants. One particular NCBT, the electroneutral Na/HCO(3)(-) cotransporter NBCn2 (SLC4A10), which is predominantly expressed in brain, has three known splice variants-NBCn2-A, -B, and -C-as well as a potential variant-D. It is important to know the tissue-specific expression of the splice variants for understanding the physiological roles of NBCn2 in central nervous system. In the present study, we developed three novel rabbit polyclonal antibodies against NBCn2: (1) anti-ABCD, which recognizes all four variants; (2) anti-BD, which recognizes NBCn2-B and -D; (3) anti-CD, which recognizes NBCn2-C and -D. By western blotting, we examined the expression and distribution of NBCn2 splice variants in five brain regions: cerebral cortex, subcortex, cerebellum, hippocampus, and medulla. The expression pattern revealed with anti-ABCD is distinct from those revealed with anti-BD and anti-CD. Moreover, by using immunoprecipitation in combination with western blotting, we demonstrate that NBCn2-D does indeed exist and that it is predominantly expressed in subcortex, to a lesser extent in medulla, but at very low levels in cortex, cerebellum, and hippocampus. NBCn2-A may be the dominant variant in mouse brain as a whole, and may also dominate in cerebral cortex, cerebellum, and hippocampus. Immunohistochemistry with anti-ABCD shows that NBCn2 is highly expressed in choroid plexus, cortex, molecular layer of cerebellum, hippocampus, and some specific regions of the brainstem., (Copyright (c) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

Regulation of sodium balance is a critical factor in the maintenance of euvolemia, and dysregulation of renal sodium excretion results in disorders of altered intravascular volume, such as hypertension. The amiloride-sensitive epithelial sodium channel (ENaC) is thought to be the only mechanism for sodium transport in the cortical collecting duct (CCD) of the kidney. However, it has been found that much of the sodium absorption in the CCD is actually amiloride insensitive and sensitive to thiazide diuretics, which also block the Na-Cl cotransporter (NCC) located in the distal convoluted tubule. In this study, we have demonstrated the presence of electroneutral, amiloride-resistant, thiazide-sensitive, transepithelial NaCl absorption in mouse CCDs, which persists even with genetic disruption of ENaC. Furthermore, hydrochlorothiazide (HCTZ) increased excretion of Na+ and Cl- in mice devoid of the thiazide target NCC, suggesting that an additional mechanism might account for this effect. Studies on isolated CCDs suggested that the parallel action of the Na+-driven Cl-/HCO3- exchanger (NDCBE/SLC4A8) and the Na+-independent Cl-/HCO3- exchanger (pendrin/SLC26A4) accounted for the electroneutral thiazide-sensitive sodium transport. Furthermore, genetic ablation of SLC4A8 abolished thiazide-sensitive NaCl transport in the CCD. These studies establish what we believe to be a novel role for NDCBE in mediating substantial Na+ reabsorption in the CCD and suggest a role for this transporter in the regulation of fluid homeostasis in mice.

The Xenopus laevis oocyte is a model system for the electrophysiological study of exogenous ion transporters. Three main reasons make the oocyte suitable for this purpose: (a) it has a large cell size (approximately 1mm diameter), (b) it has an established capacity to produce-from microinjected mRNAs or cRNAs-exogenous ion transporters with close-to-physiological post-translational modifications and actions, and (c) its membranes contain endogenous ion-transport activities which are usually smaller in magnitude than the activities of exogenously-expressed ion transporters. The expression of ion transporters as green fluorescent protein fusions allows the fluorometric assay of transporter yield in living oocytes. Monitoring of transporter-mediated movement of ions such as Cl(-), H(+) (and hence base equivalents like OH(-) and HCO(3)(-)), K(+), and Na(+) is achieved by positioning the tips of ion-sensitive microelectrodes inside the oocyte and/or at the surface of the oocyte plasma membrane. The use of ion-sensitive electrodes is critical for studying net ion-movements mediated by electroneutral transporters. The combined use of fluorometry and electrophysiology expedites transporter study by allowing measurement of transporter yield prior to electrophysiological study and correlation of relative transporter yield with transport rates.

V3526, a genetically modified strain of Venezuelan equine encephalitis virus (VEEV), was formalin inactivated for evaluation as a next generation vaccine candidate for VEEV. In this study, we tested formalin-inactivated V3526 (fV3526) with and without adjuvant for immunogenicity and efficacy in BALB/c mice and results were compared to the existing inactivated VEEV vaccine, C84. Mice were vaccinated intramuscularly (IM) or subcutaneously (SC) with fV3526 formulations and challenged with VEEV IAB Trinidad donkey (VEEV TrD) strain by SC or aerosol exposure. Efficacy following SC or aerosol challenge was not significantly different between the fV3526 formulations or compared to C84 despite C84 being administered in more doses and higher concentration of viral protein per dose. These data support further evaluation of fV3526 formulations as a next generation VEEV vaccine., (Copyright 2010 Elsevier Ltd. All rights reserved.)

A multisystem approach was used to assess the efficiency of several methods for inactivation of Venezuelan equine encephalitis virus (VEEV) vaccine candidates. A combination of diverse assays (plaque, in vitro cytopathology and mouse neurovirulence) was used to verify virus inactivation, along with the use of a specific ELISA to measure retention of VEEV envelope glycoprotein epitopes in the development of several inactivated VEEV candidate vaccines derived from an attenuated strain of VEEV (V3526). Incubation of V3526 aliquots at temperatures in excess of 64 degrees C for periods >30 min inactivated the virus, but substantially reduced VEEV specific monoclonal antibody binding of the inactivated material. In contrast, V3526 treated either with formalin at concentrations of 0.1% or 0.5% (v/v) for 4 or 24 h, or irradiated with 50 kGy gamma radiation rendered the virus non-infectious while retaining significant levels of monoclonal antibody binding. Loss of infectivity of both the formalin inactivated (fV3526) and gamma irradiated (gV3526) preparations was confirmed via five successive blind passages on BHK-21 cells. Similarly, loss of neurovirulence for fV3526 and gV3526 was demonstrated via intracerebral inoculation of suckling BALB/c mice. Excellent protection against subcutaneous challenge with VEEV IA/B Trinidad donkey strain was demonstrated using a two dose immunization regimen with either fV3526 or gV3526. The combination of in vitro and in vivo assays provides a practical approach to optimize manufacturing process parameters for development of other inactivated viral vaccines., (2009 Elsevier B.V. All rights reserved.)

We recently developed a gamma-irradiation method to inactivate V3526, a live-attenuated Venezuelan equine encephalitis virus (VEEV) vaccine candidate. Dosage and schedule studies were conducted to evaluate the immunogenicity and efficacy of gamma-irradiated V3526 (gV3526). Subcutaneous (SC) and low dosage intramuscular (IM) administration of gV3526 were highly effective in protecting mice against a SC challenge with VEEV IA/B Trinidad Donkey strain, but not against an equivalent aerosol challenge. More robust immune responses and increased protective efficacy were noted when the IM dosage of gV3526 was increased. IM administration of gV3526 formulated with either CpG or CpG plus Alhydrogel further augmented the immune response in mice and resulted in 100% protection against aerosol challenge.

Non-human primates (NHP) are considered to be the most appropriate model for predicting how humans will respond to many infectious diseases. Due to ethical and monetary concerns associated with the use of NHP, rodent models that are as predictive of responses likely to be seen in human vaccine recipients are warranted. Using implanted telemetry devices, body temperature and activity were monitored in inbred and outbred mouse strains following administration of the live-attenuated vaccine for Venezuelan equine encephalitis virus (VEEV), V3526. Following analysis of individual mouse data, only outbred mouse strains showed changes in diurnal temperature and activity profiles following vaccination. Similar changes were observed following VEEV challenge of vaccinated outbred mice. From these studies, we conclude, outbred mouse strains implanted with telemeters are a sensitive model for predicting responses in humans following vaccination.

We employed directed molecular evolution to improve the cross-reactivity and immunogenicity of the Venezuelan equine encephalitis virus (VEEV) envelope glycoproteins. The DNA encoding the E1 and E2 proteins from VEEV subtypes IA/B and IE, Mucambo virus (MUCV), and eastern and western equine encephalitis viruses (EEEV and WEEV) were recombined in vitro to create libraries of chimeric genes expressing variant envelope proteins. ELISAs specific for all five parent viruses were used in high-throughput screening to identify those recombinant DNAs that demonstrated cross-reactivity to VEEV, MUCV, EEEV, and WEEV after administration as plasmid vaccines in mice. Selected variants were then used to vaccinate larger cohorts of mice and their sera were assayed by both ELISA and by plaque reduction neutralization test (PRNT). Representative variants from a library in which the E1 gene from VEEV IA/B was held constant and only the E2 genes of the five parent viruses were recombined elicited significantly increased neutralizing antibody titers to VEEV IA/B compared to the parent DNA vaccine and provided improved protection against aerosol VEEV IA/B challenge. Our results indicate that it is possible to improve the immunogenicity and protective efficacy of alphavirus DNA vaccines using directed molecular evolution.

Mammalian genomes contain 10 SLC4 genes that, between them, encode three Cl-HCO(3) exchangers, five Na(+)-coupled HCO(3) transporters (NCBTs), one reported borate transporter, and what is reported to be a fourth Cl-HCO(3) exchanger. The NCBTs are expressed throughout the body and play important roles in maintaining intracellular and whole-body pH, as well as contributing to transepithelial transport processes. The importance of NCBTs is underscored by the genetic association of dysfunctional NCBT genes with blindness, deafness, epilepsy, hypertension and metal retardation. Key to understanding the action and regulation of NCBTs is an appreciation of the diversity of NCBT gene products. The transmembrane domains of human NCBT paralogs are 50-84% identical to each other at the amino acid level, and are capable of a diverse range of actions, including electrogenic Na/HCO(3) cotransport (i.e. NBCe1 and NBCe2) and electroneutral Na/HCO(3) cotransport (i.e. NBCn1 and NBCn2), as well as Na(+)-dependent Cl-HCO(3) exchange (i.e. NDCBE). Furthermore, by the use of alternative promoters and alternative-splicing events, individual SLC4 genes have the potential to generate multiple splice variants (as many as 16 in the case of NBCn1), each of which could have unique temporal and spatial patterns of distribution, unitary transporter activity (i.e. flux mediated by one molecule), array of protein-binding partners, and complement of regulatory stimuli. In the first section of this review, we summarize our present knowledge of the function and distribution of mammalian NCBTs and their multiple variants. In the second section of this review we consider the molecular consequences of NCBT variation.

Background: In clinical transplantation, ischemia-reperfusion injury (I/RI) causes damage to DNA. We hypothesize that one form of damage is the demethylation of methylated cytosines in the donor genome caused by the oxidative environment created first by ischemia, and subsequently by reperfusion on transplantation. This study contributes to the understanding of how the short-lived and transient ischemic insult may influence chronic pathological changes that occur in clinical transplantation in the long term., Methods: A model of I/RI and chronic rejection; Fisher to Fisher kidney transplant rendered cold-ischemic for 4 hr before transplantation, to induce antigen-independent chronic nephropathy over a 6-month period, was used. Tissue was assessed by histopathology and methylation by pyrosequencing analysis., Results: An epigenetic map of the rat renal C3 promoter was produced, which identified methylated Cytosine phospho Guanine (CpG) sites coincident to cytokine response elements and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB) binding sites. Pyrosequencing analysis showed that the tissue that had undergone 4 hr ischemia and reperfusion developed aberrant demethylation of cytosines in putative regulatory sites within the C3 promoter., Conclusion: These findings may describe a newly recognized phenomena in the field of transplantation. Aberrant demethylation has long been linked to the development of tumors, and our data suggest a similar mechanism of gene dysregulation that may be initiated by I/RI with acute and chronic effects. These data may contribute to a further understanding of how the short lived and transient ischemic insult influences chronic pathological changes that occur even in the absence of major histocompatibility complex disparity in transplantation.

The reported sequences of the human and mouse Na+-driven Cl-/HCO3(-) exchangers (NDCBEs) differ greatly in their extreme cytosolic COOH termini (Ct). In human NDCBE (NDCBE-B), a 17-amino acid (aa) sequence replaces 66 aa at the equivalent position in mouse NDCBE (NDCBE-A). We performed 5'- and 3'-rapid amplification of cDNA ends (RACE) on human brain cDNA, followed by PCR of full-length cDNAs to determine whether the human SLC4A8 gene was capable of producing the mouselike Ct sequence. Our study confirmed the presence in human cDNA of mouse NDCBE-like transcripts (human NDCBE-A) and also disclosed the existence of three further novel NDCBE transcripts that we have called NDCBE-C, NDCBE-D, and NDCBE-D'. The novel NDCBE-C/D/D' transcripts initiate at a novel "exon 0" positioned approximately 35 kb upstream of the first exon of NDCBE-A/B. NDCBE-C/D/D' protein products are predicted to be truncated by 54 aa in the cytosolic NH(2) terminus (Nt) compared with NDCBE-A/B. Our data, combined with a new in silico analysis of partial transcripts reported by others in the region of the human SLC4A8 gene, increase the known extent of the SLC4A8 gene by 49 kb, to 124 kb. A functional comparison of NDCBE-A/B/C/D expressed in Xenopus oocytes demonstrates that the Nt variation does not affect the basal functional expression of NDCBE, but those with the shorter Ct have a 25-50% reduced functional expression compared with those with the longer Ct. By comparison with an artificially truncated NDCBE that contains neither 17-aa nor 66-aa Ct cassette, we determined that the functional difference is unrelated to the 66-aa cassette of NDCBE-A/C, but is instead due to an inhibitory effect of the 17-aa cassette of NDCBE-B/D.

The SLC4A10 gene product, commonly known as NCBE, is highly expressed in rodent brain and has been characterized by others as a Na(+)-driven Cl-HCO(3) exchanger. However, some of the earlier data are not consistent with Na(+)-driven Cl-HCO(3) exchange activity. In the present study, northern blot analysis showed that, also in humans, NCBE transcripts are predominantly expressed in brain. In some human NCBE transcripts, splice cassettes A and/or B, originally reported in rats and mice, are spliced out. In brain cDNA, we found evidence of a unique partial splice of cassette B that is predicted to produce an NCBE protein with a novel C terminus containing a protein kinase C phosphorylation site. We used pH-sensitive microelectrodes to study the molecular physiology of human NCBE expressed in Xenopus oocytes. In agreement with others we found that NCBE mediates the 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid-sensitive, Na(+)-dependent transport of HCO(3)(-). For the first time, we demonstrated that this transport process is electroneutral. Using Cl(-)-sensitive microelectrodes positioned at the oocyte surface, we found that, unlike both human and squid Na(+)-driven Cl-HCO(3) exchangers, human NCBE does not normally couple the net influx of HCO(3)(-) to a net efflux of Cl(-). Moreover we found that that the (36)Cl efflux from NCBE-expressing oocytes, interpreted by others to be coupled to the influx of Na(+) and HCO(3)(-), actually represents a CO(2)/HCO(3)(-)-stimulated Cl(-) self-exchange not coupled to either Na(+) or net HCO(3)(-) transport. We propose to rename NCBE as the second electroneutral Na/HCO(3) cotransporter, NBCn2.

The Na(+)-driven Cl-HCO(3) exchanger (NDCBE or SLC4A8) is a member of the solute carrier 4 (SLC4) family of HCO(3)(-) transporters, which includes products of 10 genes with similar sequences. Most SLC4 members play important roles in regulating intracellular pH (pH(i)). Physiological studies suggest that NDCBE is a major pH(i) regulator in at least hippocampal (HC) pyramidal neurons. We generated a polyclonal rabbit antibody directed against the first 18 residues of the cytoplasmic N terminus (Nt) of human NDCBE. By Western blotting, the antibody distinguishes NDCBE-as a purified Nt peptide or a full-length transporter (expressed in Xenopus oocytes)-from other Na(+)-coupled HCO(3)(-) transporters. By Western blotting, the antiserum recognizes an approximately 135-kDa band in several brain regions of adult mice: the cerebral cortex (CX), subcortex (SCX), cerebellum (CB), and HC. In CX, PNGase F treatment reduces the molecular weight to approximately 116 kDa. By immunocytochemistry, affinity-purified (AP) NDCBE antibody stains the plasma membrane of neuron cell bodies and processes of rat HC neurons in primary culture as well as freshly dissociated mouse HC neurons. The AP antibody does not detect substantial NDCBE levels in freshly dissociated HC astrocytes, or astrocytes in HC or CB sections. By immunohistochemistry, the AP antibody recognizes high levels of NDCBE in neurons of CX, HC (including pyramidal neurons in Cornu Ammonis (CA)1-3 and dentate gyrus), substantial nigra, medulla, cerebellum (especially Purkinje and granular cells), and the basolateral membrane of fetal choroid plexus. Thus, NDCBE is in a position to contribute substantially to pH(i) regulation in multiple CNS neurons.

An ERG gene 'break-apart' fluorescence in situ hybridization (FISH) assay has been used to screen whole-mount prostatectomy specimens for rearrangements at the ERG locus. In cancers containing ERG alterations the observed pattern of changes was often complex. Different categories of ERG gene alteration were found either together in a single cancerous region or within separate foci of cancer in the same prostate slice. In some cases the juxtaposition of particular patterns of ERG alterations suggested possible mechanisms of tumour progression. Prostates harbouring ERG alterations commonly also contained cancer that lacked rearrangements of the ERG gene. A single trans-urethral resection of the prostate specimen examined harboured both ERG and ETV1 gene rearrangements demonstrating that the observed complexity may, at least in part, be explained by multiple ETS gene alterations arising independently in a single prostate. In a search for possible precursor lesions clonal ERG rearrangements were found both in high grade prostatic intraepithelial neoplasia (PIN) and in atypical in situ epithelial lesions consistent with the diagnosis of low grade PIN. Our observations support the view that ERG gene alterations represent an initiating event that promotes clonal expansion initially to form regions of epithelial atypia. The complex patterns of ERG alteration found in prostatectomy specimens have important implications for the design of experiments investigating the clinical significance and mechanism of development of individual prostate cancers.

In an attempt to improve the current live, attenuated vaccine (TC-83) for Venezuelan equine encephalitis virus (VEEV), specific mutations associated with attenuation of VEEV in rodent models were inserted into a full-length cDNA clone of the Trinidad donkey strain of VEEV by site-directed mutagenesis. Because some viruses have been reported to be more pathogenic when introduced by mosquito bite than the same virus introduced by needle inoculation, there were concerns that the presence of mosquito saliva, or changes in the virus caused by replication in a mosquito, might allow the virus to overcome the protective effects of prior vaccination with V3526. Therefore, we determined if hamsters vaccinated with V3526 were protected from challenge with the virulent Trinidad donkey strain of VEEV. All non-vaccinated hamsters died after intraperitoneal challenge or after being fed on by VEEV-inoculated Aedes taeniorhynchus. In contrast, hamsters vaccinated with V3526 were resistant to intraperitoneal challenge and infection by VEEV-infected Ae. taeniorhynchus. Therefore, the V3526 candidate vaccine elicits protection against VEEV infection by mosquito bite.

NCBE (SLC4A10) is a member of the SLC4 family of bicarbonate transporters, several of which play important roles in intracellular-pH regulation and transepithelial HCO(3)(-) transport. Here we characterize a new antibody that was generated in rabbit against a fusion protein consisting of maltose-binding protein and the first 135 amino acids (aa) of the N-terminus of human NCBE. Western blotting--both of purified peptides representing the initial approximately 120 aa of the transporters and of full-length transporters expressed in Xenopus oocytes--demonstrated that the antibody is specific for NCBE versus the two most closely related proteins, NDCBE (SLC4A8) and NBCn1 (SLC4A7). Western blotting of tissue in four regions of adult mouse brain indicates that NCBE is expressed most abundantly in cerebral cortex (CX), cerebellum (CB) and hippocampus (HC), and less so in subcortex (SCX). NCBE protein was present in CX, CB, and HC microdissected to avoid choroid plexus. Immunocytochemistry shows that NCBE is present at the basolateral membrane of embryonic day 18 (E18) fetal and adult choroid plexus. NCBE protein is present by Western blot and immunocytochemistry in cultured and freshly dissociated HC neurons but not astrocytes. By Western blot, nearly all NCBE in mouse and rat brain is highly N-glycosylated (approximately 150 kDa). PNGase F reduces the molecular weight (MW) of natural NCBE in mouse brain or human NCBE expressed in oocytes to approximately the predicted MW of the unglycosylated protein. In oocytes, mutating any one of the three consensus N-glycosylation sites reduces glycosylation of the other two, and the triple mutant exhibits negligible functional expression.

Human red cell anion exchanger AE1 (band 3) is an electroneutral Cl-HCO3- exchanger with 12-14 transmembrane spans (TMs). Previous work using Xenopus oocytes has shown that two co-expressed fragments of AE1 lacking TMs 6 and 7 are capable of forming a stilbene disulphonate-sensitive (36)Cl-influx pathway, reminiscent of intact AE1. In the present study, we create a single construct, AE1Delta(6: 7), representing the intact protein lacking TMs 6 and 7. We expressed this construct in Xenopus oocytes and evaluated it employing a combination of two-electrode voltage clamp and pH-sensitive microelectrodes. We found that, whereas AE1Delta(6: 7) has some electroneutral Cl-base exchange activity, the protein also forms a novel anion-conductive pathway that is blocked by DIDS. The mutation Lys(539)Ala at the covalent DIDS-reaction site of AE1 reduced the DIDS sensitivity, demonstrating that (1) the conductive pathway is intrinsic to AE1Delta(6: 7) and (2) the conductive pathway has some commonality with the electroneutral anion-exchange pathway. The conductance has an anion-permeability sequence: NO3- approximately I- > NO2- > Br- > Cl- > SO4(2-) approximately HCO3- approximately gluconate- approximately aspartate- approximately cyclamate-. It may also have a limited permeability to Na+ and the zwitterion taurine. Although this conductive pathway is not a usual feature of intact mammalian AE1, it shares many properties with the anion-conductive pathways intrinsic to two other Cl-HCO3- exchangers, trout AE1 and mammalian SLC26A7.

The primary purpose of this investigation was to study oxidative demethylation of DNA following ischemia/reperfusion injury (I/RI) that putatively influences posttransplant gene expression in transplanted kidneys. Our hypothesis was that as a result of I/RI, oxidative damage, which is inherent in solid organ transplantation, may lead to aberrant demethylation of cytosine-guanine (CpG) sites within gene promoter regions of DNA. The methylated CpG sites normally contribute to the binding of proteins that render DNA inaccessible to transcription factors. Therefore, conversion of methylated cytosines to nonmethylated cytosines by oxidative damage in postischemic organs might facilitate enhanced gene expression in donor organs by exposing the demethylated CpG site in a gene promoter to DNA-binding proteins that enhance gene transcription. In this study, we investigated the demethylation of a specific CpG within the IFNgamma response element resident in the promoter region of the C3 gene in the rat kidney. In response to 24 hours of cold ischemia and a subsequent 2 hours of reperfusion in an isolated ex-vivo circuit, we observed a significant change in the ratio of methylated to unmethylated cytosines at this site. Epigenetic modifications to donor DNA have not been previously investigated, but our own data suggests that they have the potential to modify gene expression posttransplantation. Since epigenetic modification may become stable and heritable upon mitosis, such changes to the donor organ DNA may persist with enormous implications for transplant outcomes.

The human electrogenic renal Na-HCO(3) cotransporter (NBCe1-A; SLC4A4) is localized to the basolateral membrane of proximal tubule cells. Mutations in the SLC4A4 gene cause an autosomal recessive proximal renal tubular acidosis (pRTA), a disease characterized by impaired ability of the proximal tubule to reabsorb HCO(3)(-) from the glomerular filtrate. Other symptoms can include mental retardation and ocular abnormalities. Recently, a novel homozygous missense mutant (R881C) of NBCe1-A was reported from a patient with a severe pRTA phenotype. The mutant protein was described as having a lower than normal activity when expressed in Xenopus oocytes, despite having normal Na(+) affinity. However, without trafficking data, it is impossible to determine the molecular basis for the phenotype. In the present study, we expressed wild-type NBCe1-A (WT) and mutant NBCe1-A (R881C), tagged at the COOH terminus with enhanced green fluorescent protein (EGFP). This approach permitted semiquantification of surface expression in individual Xenopus oocytes before assay by two-electrode voltage clamp or measurements of intracellular pH. These data show that the mutation reduces the surface expression rather than the activity of the individual protein molecules. Confocal microscopy on polarized mammalian epithelial kidney cells [Madin-Darby canine kidney (MDCK)I] expressing nontagged WT or R881C demonstrates that WT is expressed at the basolateral membrane of these cells, whereas R881C is retained in the endoplasmic reticulum. In summary, the pathophysiology of pRTA caused by the R881C mutation is likely due to a deficit of NBCe1-A at the proximal tubule basolateral membrane, rather than a defect in the transport activity of individual molecules.

Others report that carbonic anhydrase II (CA II) binds to the C termini of the anion exchanger AE1 and the electrogenic Na/HCO3 cotransporter NBCe1-A, enhancing transport. After injecting oocytes with NBCe1-A cRNA (Day 0), we measured NBC current (I(NBC)) by two-electrode voltage clamp (Day 3), injected CA II protein + Tris or just Tris (Day 3), measured I(NBC) or the initial rate at which the intracellular pH fell (dpH(i)/dt) upon applying 5% CO2 (Day 4), exposed oocytes to the permeant CA inhibitor ethoxzolamide (EZA), and measured I(NBC) or dpH(i)/dt (Day 4). Because dpH(i)/dt was greater in CA II than Tris oocytes, and EZA eliminated the difference, injected CA II was functional. I(NBC) slope conductance was unaffected by injecting CA II. Moreover, EZA had identical effects in CA II versus Tris oocytes. Thus, injected CA II does not enhance NBC activity. In a second protocol, we made a fusion protein with enhanced green fluorescent protein (EGFP) at the 5' end of NBCe1-A and CA II at the 3' end (EGFP-e1-CAII). We measured I(NBC) or dpH(i)/dt (days 3-4), exposed oocytes to EZA, and measured I(NBC) or dpH(i)/dt (Day 3-4). dpH(i)/dt was greater in oocytes expressing EGFP-e1-CA II versus EGFP-e1, and EZA eliminated the difference. Thus, fused CA II was functional. Slope conductances of EGFP-e1-CAII versus EGFP-e1 oocytes were indistinguishable, and EZA had no effect. Thus, even when fused to NBCe1-A, CA II does not enhance NBCe1-A activity.

Cynomolgus macaques were exposed by aerosol to a virulent strain of western equine encephalitis virus (WEEV). Between 4 and 6 days after exposure, macaques had a significantly elevated temperature that lasted for 3-4 days. Clinical signs of encephalitis began as the body temperature decreased, and then they rapidly increased in severity. Cynomolgus macaques with clinical signs of encephalitis had elevated white cell counts in the blood caused mostly by increased numbers of segmented neutrophils and monocytes. Elevated serum glucose levels also correlated with the severity of the clinical signs of encephalitis. Three cynomolgus macaques died; immunohistochemical evidence of viral antigen was present in the brain and central nervous system (CNS). Microscopic analysis also revealed a marked lymphocytic infiltrate in the CNS. Cynomolgus macaques will serve as a useful model of aerosol exposure to WEEV for the evaluation of potential vaccine candidates.

The heightened concerns about bioterrorism and the use of biowarfare agents have prompted substantial increased efforts towards the development of vaccines against a wide range of organisms, toxins, and viruses. An increasing variety of platforms and strategies have been analyzed for their potential as vaccines against these agents. DNA vectors, live-attenuated viruses and bacteria, recombinant proteins combined with adjuvant, and viral- or bacterial-vectored vaccines have been developed as countermeasures against many potential agents of bioterrorism or biowarfare. The use of viruses, for example adenovirus, vaccinia virus, and Venezuelan equine encephalitis virus, as vaccine vectors has enabled researchers to develop effective means for countering the threat of bioterrorism and biowarfare. An overview of the different viral vectors and the threats they counter will be discussed.

Two live, attenuated strains of Venezuelan equine encephalitis virus (VEE), IE1150K and V3526, were administered to macaques to determine if they could elicit protection against an aerosol challenge with virulent VEE virus of the IE variety (VEEV-IE). These viruses were rescued from full-length cDNA clones of 68U201 (VEEV-IE variety) and Trinidad donkey (VEEV-IA/B variety), respectively, and both have a furin cleavage site deletion mutation and a second-site resuscitating mutation. Both vaccines elicited neutralizing antibodies to viruses of the homologous variety but not to viruses of the heterologous variety. Eight weeks after vaccination, the macaques were challenged by aerosol exposure to virulent 68U201. Macaques vaccinated with V3526 were protected as well as macaques inoculated with IE1009, the wild-type infectious clone of 68U201. However, IE1150K failed to significantly protect macaques relative to controls. V3526 has now been shown to protect macaques against both IA/B [Pratt WD, Davis NL, Johnston RE, Smith JF. Genetically engineered, live attenuated vaccines for Venezuelan equine encephalitis: testing in animal models. Vaccine 2003;21(25-26):3854-62] and IE strains of VEE viruses.

To elucidate the pathogenesis of eastern equine encephalitis (EEE) virus infections, we used histopathology, immunohistochemistry, and in situ hybridization to track the spread and early cellular targets of viral infection in mice. Young mice were inoculated with virulent EEE virus in their right rear footpad and were followed in a time-course study for 4 days. Virulent EEE virus produced a biphasic illness characterized by an early self-limiting replication phase in peripheral tissues followed by an invariably fatal central nervous system (CNS) phase. In the early extraneural phase, there was primary amplifying replication of virus within fibroblasts at the inoculation site and within osteoblasts in active growth areas of bone that resulted in a transient high-titer viremia. Pathological changes and viral infection were observed as early as 12 hours post-infection (PI) in osteoblasts, skeletal muscle myocytes, and in fibroblasts along fascial sheaths. The severity and extent of infection in peripheral tissues peaked at day 1 PI. In the neural phase of infection, virus was first detected in the brain on day 1 PI, with rapid interneuronal spread of infection leading to death by day 4 PI. EEE virus appeared to be directly cytopathic for neurons. The very rapid onset and apparently random and widely dispersed infection in the CNS, with concurrent sparing of olfactory neuroepithelium, strongly suggests that invasion of the CNS by EEE occurs by a vascular route, rather than via peripheral nerves or the olfactory neuroepithelium. Our finding that metaphyseal osteoblasts are an early site of amplifying viral replication may explain the higher-titer viremias and higher incidence of neuroinvasion and fulminant encephalitis seen in the young, and may also explain why mature animals become refractory to encephalitis after peripheral inoculation with EEE virus.

The red cell anion exchanger (band 3; AE1) is a multispanning membrane protein that traverses the bilayer up to 14 times and mediates the stilbene-disulfonate-sensitive, electroneutral exchange of chloride and bicarbonate. Previous studies showed that the integrity of the third extracellular loop (EC3) of the protein was not essential for stilbene-disulfonate-sensitive chloride uptake. Here we demonstrate that the chloride uptake mediated by assemblies separated at EC3 represents the physiological electroneutral Cl(-)/HCO(3)(-) activity associated with intact AE1 protein. This provides further evidence that the 1:5 and 6:14 regions of the protein form discrete folding domains and confirms that the third extracellular loop does not play a pivotal role in AE1 transport function.

Because Venezuelan equine encephalitis viruses (VEEVs) are infectious by aerosol, they are considered to be a biological-weapons threat. Nonhuman-primate models are needed to evaluate the efficacy of candidate vaccines. In the present study, cynomolgus macaques, after aerosol exposure to either VEEV-IE or VEEV-IIIA, developed fever, viremia, and lymphopenia; the severity of the fever response, viremia, and lymphopenia correlated with the inhaled dose of VEEV. Of the 10 macaques in our study, 7 developed clinical signs indicative of encephalitis, including loss of balance and hypothermia. In the macaque, the enzootic strains used are infectious by aerosol and lead to disease, including clinical encephalitis.

Specific mutations associated with attenuation of Venezuelan equine encephalitis (VEE) virus in rodent models were identified during efforts to develop an improved VEE vaccine. Analogous mutations were produced in full-length cDNA clones of the Cba 87 strain of western equine encephalitis (WEE) virus by site-directed mutagenesis in an attempt to develop an improved WEE vaccine. Isogenic viral strains with these mutations were recovered after transfection of baby hamster kidney cells with infectious RNA. We evaluated two of these strains (WE2102 and WE2130) for their ability to replicate in and be transmitted by Culex tarsalis, the principal natural vector of WEE virus in the United States. Each of the vaccine candidates contained a deletion of the PE2 furin cleavage site and a secondary mutation in the E1 or E2 glycoprotein. Both of these potential candidates replicated in mosquitoes significantly less efficiently than did either wild-type WEE (Cba 87) virus or the parental clone (WE2000). Likewise, after intrathoracic inoculation, mosquitoes transmitted the vaccine candidate strains significantly less efficiently than they transmitted either the wild-type or the parental clone. One-day-old chickens vaccinated with either of the two vaccine candidates did not become viremic when challenged with virulent WEE virus two weeks later. Mutations that result in less efficient replication in or transmission by mosquitoes should enhance vaccine safety and reduce the possibility of accidental introduction of the vaccine strain to unintentional hosts.

Chimeric cDNA clones, pMWE1000 and pMWE2000, differing by five nucleotides at their 5' termini, were constructed of the 5' two-thirds of the western equine encephalitis (WEE) virus genome (encoding nonstructural proteins) and the 3' one-third of the eastern equine encephalitis (EEE) virus genome (encoding structural proteins). The WEE virus sequences were derived from full-length cDNA clones, pWE1000 and pWE2000, which were isogenic except for five nucleotide differences at their 5' termini and were responsible for significant differences in mouse virulence. Each cDNA clone was placed downstream from a T7 promoter to allow in vitro transcription of full-length RNA. Transfection of BHK-21 cells with the chimeric RNA by electroporation gave rise to high-titer infectious virus. The in vitro characteristics of each chimera virus were determined by electrophoretic analysis of its structural proteins, plaque morphology, neutralization characteristics, replication kinetics, and rate of viral RNA synthesis. With the exception of plaque morphology, the in vitro characteristics of MWE1000 and MWE2000 were indistinguishable from the parental EEE virus. Subcutaneous inoculation of 5-week-old C57BL/6 mice with varying doses of MWE1000 or MWE2000 virus demonstrated that both chimeric viruses were significantly attenuated compared to the parental WEE virus (Cba 87) and EEE virus (PE-6). Animals infected with 10(5) PFU or more of either MWE1000 or MWE2000 were completely protected from lethal challenge with the virulent EEE virus, FL91-4679, but were not protected from virulent WEE virus Cba 87 challenge. Construction of viable virus chimeras often results in attenuated viruses that may hold promise as genetically engineered alphavirus vaccine candidates (R. J. Kuhn, D. E. Griffin, K. E. Owen, H. G. M. Niesters, and J. H. Strauss, 1996, J. Virol. 70, 7900-7909).

A yearling quarter horse, which was raised in southern California, received routine vaccinations for prevention of infection by Eastern equine encephalomyelitis virus (EEEV). One week later, severe neurologic signs developed, and the horse was humanely destroyed. A vaccine-related encephalomyelitis was later suspected. A final diagnosis of EEEV infection was established on the basis of acute onset of the neurologic signs, histopathologic and serologic testing, and isolation and molecular characterization of EEEV from brain tissue. The vaccine was extensively tested for viral inactivation. Nucleotide sequences from the vaccine and the virus isolated in the affected horse were also compared. In California, arboviral encephalomyelitides are rarely reported, and EEEV infection has not previously been documented. This report describes the occurrence of EEEV infection in the horse and the investigation to determine the source of infection, which was not definitively identified.

A candidate vaccine against botulinum neurotoxin serotype A (BoNT/A) was developed by using a Venezuelan equine encephalitis (VEE) virus replicon vector. This vaccine vector is composed of a self-replicating RNA containing all of the VEE nonstructural genes and cis-acting elements and also a heterologous immunogen gene placed downstream of the subgenomic 26S promoter in place of the viral structural genes. In this study, the nontoxic 50-kDa carboxy-terminal fragment (H(C)) of the BoNT/A heavy chain was cloned into the replicon vector (H(C)-replicon). Cotransfection of BHK cells in vitro with the H(C)-replicon and two helper RNA molecules, the latter encoding all of the VEE structural proteins, resulted in the assembly and release of propagation-deficient, H(C) VEE replicon particles (H(C)-VRP). Cells infected with H(C)-VRP efficiently expressed this protein when analyzed by either immunofluorescence or by Western blot. To evaluate the immunogenicity of H(C)-VRP, mice were vaccinated with various doses of H(C)-VRP at different intervals. Mice inoculated subcutaneously with H(C)-VRP were protected from an intraperitoneal challenge of up to 100,000 50% lethal dose units of BoNT/A. Protection correlated directly with serum enzyme-linked immunosorbent assay titers to BoNT/A. The duration of the immunity achieved was tested at 6 months and at 1 year postvaccination, and mice challenged at these times remained refractory to challenge with BoNT/A.