The light chain of antibodies specific to the V2 region of HIV-1 can determine their function.

We studied the contribution of the light chain to functions of human monoclonal antibodies (mAbs) by measuring the relationships between the rate of mutations and cross-reactivity, binding affinity and neutralization activity. We analyzed 12 mAbs of two clonal families specific to the V2 region of HIV-1 derived from two chronically HIV-1 infected individuals. The clonal mAbs exhibited a range of reactivities, and the clones with superior properties were associated with the rate of mutations and the presence of particular mutated residues in the light chains, but not in the heavy chains. Our observations suggest that for some antibodies, the light chains play a vital role in antibody evolution toward more efficient ones and also suggest the importance of optimal residues rather than the rate of mutations in the variable fragment of the antibody.

A large repertoire of B cell lineages targeting one cluster of epitopes in a vaccinated rhesus macaque.

The repertoire of antibodies (Abs) produced upon vaccination against a particular antigenic site is rarely studied due to the complexity of the immunogens. We received such an opportunity when one rhesus macaque was immunized six times at 0, 4, 10, 16, 32, and 143 weeks with C4-447 peptide containing the 8-mer epitope for human monoclonal Ab (mAb) 447-52D specific to the V3 region of gp120 HIV-1. Strong anti-V3 antibody responses reached 50% binding titer in serum of 10-5 at week 10 that declined to 10-3 by week 70. After an additional boost of C4-447 peptide at week 143, titers rebounded to 10-5 at week 146, or 2.7 years after the first immunization. Using the blood sample at week 146, we produced 41 V3-specific recombinant mAbs by single B cell isolation and cloning. Sequence analysis revealed 21B cell lineages, single and clonally related, based on immunoglobulin gene usage and CDR3s. The broad repertoire of Abs directed to a small antigenic site shows the targeting potency of a vaccine-elicited immune response in rhesus macaques.

Virus Control in Vaccinated Rhesus Macaques Is Associated with Neutralizing and Capturing Antibodies against the SHIV Challenge Virus but Not with V1V2 Vaccine-Induced Anti-V2 Antibodies Alone.

The role of vaccine-induced anti-V2 Abs was tested in three protection experiments in rhesus macaques. In an experiment using immunogens similar to those in the RV144 vaccine trial (Anti-envelope [Env]), nine rhesus macaques were coimmunized with gp16092TH023 DNA and SIV gag and gp120A244 and gp120MN proteins. In two V2-focused experiments (Anti-V2 and Anti-V2 Mucosal), nine macaques in each group were immunized with V1V292TH023 DNA, V1V2A244 and V1V2CasaeA2 proteins, and cyclic V2CaseA2 peptide. DNA and protein immunogens, formulated in Adjuplex, were given at 0, 4, 12, and 20 weeks, followed by intrarectal SHIVBaL.P4 challenges. Peak plasma viral loads (PVL) of 106-107 copies/ml developed in all nine sham controls. Overall, PVL was undetectable in one third of immunized macaques, and two animals tightly controlled the virus with the Anti-V2 Mucosal vaccine strategy. In the Anti-Env study, Abs that captured or neutralized SHIVBaL.P4 inversely correlated with PVL. Conversely, no correlation with PVL was found in the Anti-V2 experiments with nonneutralizing plasma Abs that only captured virus weakly. Titers of Abs against eight V1V2 scaffolds and cyclic V2 peptides were comparable between controllers and noncontrollers as were Ab-dependent cellular cytotoxicity and Ab-dependent cell-mediated virus inhibition activities against SHIV-infected target cells and phagocytosis of gp120-coated beads. The Anti-Env experiment supports the role of vaccine-elicited neutralizing and nonneutralizing Abs in control of PVL. However, the two V2-focused experiments did not support a role for nonneutralizing V2 Abs alone in controlling PVL, as neither Ab-dependent cellular cytotoxicity, Ab-dependent cell-mediated virus inhibition, nor phagocytosis correlated inversely with heterologous SHIVBaL.P4 infection.

Anti-V2 antibody deficiency in individuals infected with HIV-1 in Cameroon.

The results of the RV144 vaccine clinical trial showed a correlation between high level of anti-V1V2 antibodies (Abs) and a decreased risk of acquiring HIV-1 infection. This turned the focus of HIV vaccine design to the induction of elevated levels of anti-V2 Abs to increase vaccine efficacy. In plasma samples from HIV-1 infected Cameroonian individuals, we observed broad variations in levels of anti-V2 Abs, and 6 of the 79 plasma samples tested longitudinally displayed substantial deficiency of V2 Abs. Sequence analysis of the V2 region from plasma viruses and multivariate analyses of V2 characteristics showed a significant difference in several features between V2-deficient and V2-reactive plasma Abs. These results suggest that HIV vaccine immunogens containing a shorter V2 region with fewer glycosylation sites and higher electrostatic charges can be beneficial for induction of a higher level of anti-V2 Abs and thus contribute to HIV vaccine efficacy.

Association of Diverse Genotypes and Phenotypes of Immune Cells and Immunoglobulins With the Course of HIV-1 Infection.

Disease progression among HIV-1-infected individuals varies widely, but the mechanisms underlying this variability remains unknown. Distinct disease outcomes are the consequences of many factors working in concert, including innate and adaptive immune responses, cell-mediated and humoral immunity, and both genetic and phenotypic factors. Current data suggest that these multifaceted aspects in infected individuals should be considered as a whole, rather than as separate unique elements, and that analyses must be performed in greater detail in order to meet the requirements of personalized medicine and guide optimal vaccine design. However, the wide adoption of antiretroviral therapy (ART) influences the implementation of systematic analyses of the HIV-1-infected population. Consequently, fewer data will be available for acquisition in the future, preventing the comprehensive investigations required to elucidate the underpinnings of variability in disease outcome. This review seeks to recapitulate the distinct genotypic and phenotypic features of the immune system, focusing in particular on comparing the surface proteins of immune cells among individuals with different HIV infection outcomes.

Major Discrepancies of HCV Viral Loads Determined by Real Time Polymerase Chain Reaction Caused by Use of Collection Tubes that Differ Only with the Presence or Absence of Serum Separator.

BACKGROUND: For viral load (VL) analysis of hepatitis C virus (HCV) by Real Time PCR, approved collection tubes were lavender top (LTT) and plasma processing (PPT) tubes, that differ in that PPTs include plasma separator. Using a Real Time PCR method, we investigated how the results correlated for the two tube types. METHODS: The plasma fractions of blood samples from each of 202 patients were collected in LTT and PPT tubes at the same time and were assayed for HCV VL by the Abbott m2000 Real Time PCR System; the results were analyzed statistically. RESULTS: VLs for 103 samples for both tubes were negative. Positive results were obtained for another 79 tube pairs, including six with VLs for LTTs but values below linearity for the corresponding PPTs. For the 73 samples for which quantitative results were obtained for both tube types, VLs were statistically higher in LTTs (means 1,817,821.8 in LTTs and 1,083,669.1 in PPTs, p=0.006, alpha=0.05) using the paired t-test and confirmed by the Chi Square and McNemars tests. CONCLUSIONS: VLs in LTTs are significantly higher and more sensitive than in PPTs, suggesting the necessity of standardization of collection tubes for HCV VLs.

Differential induction of anti-V3 crown antibodies with cradle- and ladle-binding modes in response to HIV-1 envelope vaccination.

The V3 loop in the HIV envelope gp120 is one of the immunogenic sites targeted by Abs. The V3 crown in particular has conserved structural elements recognized by cross-reactive neutralizing Abs, indicating its potential contribution in protection against HIV. Crystallographic analyses of anti-V3 crown mAbs in complex with the V3 peptides have revealed that these mAbs recognize the conserved sites on the V3 crown via two distinct strategies: a cradle-binding mode (V3C) and a ladle-binding (V3L) mode. However, almost all of the anti-V3 crown mAbs studied in the past were isolated from chronically HIV-infected individuals. The extents to which the two types of anti-V3 crown Abs are generated by vaccination are unknown. This study analyzed the prevalence of V3C-type and V3L-type Ab responses in HIV-infected individuals and in HIV envelope-immunized humans and animals using peptide mimotopes that distinguish the two Ab types. The results show that both V3L-type and V3C-type Abs were generated by the vast majority of chronically HIV-infected humans, although the V3L-type were more prevalent. In contrast, only one of the two V3 Ab types was elicited in vaccinated humans or animal models, irrespective of HIV-1 envelope clades, envelope constructs (oligomeric or monomeric), and protocols (DNA plus protein or protein alone) used for vaccinations. The V3C-type Abs were produced by vaccinated humans, macaques, and rabbits, whereas the V3L-type Abs were made by mice. The V3C-type and V3L-type Abs generated by the vaccinations were able to mediate virus neutralization. These data indicate the restricted repertoires and the species-specific differences in the functional V3-specific Ab responses induced by the HIV envelope vaccines. The study implies the need for improving immunogen designs and vaccination strategies to broaden the diversity of Abs in order to target the different conserved epitopes in the V3 loop and, by extension, in the entire HIV envelope.

Monoclonal Antibodies Specific for the V2, V3, CD4-Binding Site, and gp41 of HIV-1 Mediate Phagocytosis in a Dose-Dependent Manner.

In light of the weak or absent neutralizing activity mediated by anti-V2 monoclonal antibodies (MAbs), we tested whether they can mediate Ab-dependent cellular phagocytosis (ADCP), which is an important element of anti-HIV-1 immunity. We tested six anti-V2 MAbs and compared them with 21 MAbs specific for V3, the CD4-binding site (CD4bs), and gp41 derived from chronically HIV-1-infected individuals and produced by hybridoma cells. ADCP activity was measured by flow cytometry using uptake by THP-1 monocytic cells of fluorescent beads coated with gp120, gp41, BG505 SOSIP.664, or BG505 DS-SOSIP.664 complexed with MAbs. The measurement of ADCP activity by the area under the curve showed significantly higher activity of anti-gp41 MAbs than of the members of the three other groups of MAbs tested using beads coated with monomeric gp41 or gp120; anti-V2 MAbs were dominant compared to anti-V3 and anti-CD4bs MAbs against clade C gp120ZM109 ADCP activity mediated by V2 and V3 MAbs was positive against stabilized DS-SOSIP.664 trimer but negligible against SOSIP.664 targets, suggesting that a closed envelope conformation better exposes the variable loops. Two IgG3 MAbs against the V2 and V3 regions displayed dominant ADCP activity compared to a panel of IgG1 MAbs. This superior ADCP activity was confirmed when two of three recombinant IgG3 anti-V2 MAbs were compared to their IgG1 counterparts. The study demonstrated dominant ADCP activity of anti-gp41 against monomers but not trimers, with some higher activity of anti-V2 MAbs than of anti-V3 and anti-CD4bs MAbs. The ability to mediate ADCP suggests a mechanism by which anti-HIV-1 envelope Abs can contribute to protective efficacy.IMPORTANCE Anti-V2 antibodies (Abs) correlated with reduced risk of HIV-1 infection in recipients of the RV144 vaccine, suggesting that they play a protective role, but a mechanism providing such protection remains to be determined. The rare and weak neutralizing activities of anti-V2 MAbs prompted us to study Fc-mediated activities. We compared anti-V2 MAbs with other MAbs specific for V3, CD4bs, and gp41 for Ab-dependent cellular phagocytosis (ADCP) activity, implicated in protective immunity. The anti-V2 MAbs displayed stronger activity than other anti-gp120 MAbs in screening against one of two gp120s and against DS-SOSIP, which mimics the native trimer. The activity of anti-gp41 MAbs was superior in targeting monomeric gp41 but was comparable to that seen against trimers, which may not adequately expose gp41 epitopes. While anti-envelope MAbs in general mediated ADCP activity, anti-V2 MAbs displayed some dominance compared to other MAbs. Our demonstration that anti-V2 MAbs mediate ADCP activity suggests a functional mechanism for their contribution to protective efficacy.

Ro52 autoantibodies arise from self-reactive progenitors in a mother of a child with neonatal lupus.

The detection of cardiac conduction defects in an 18-24 week old foetus in the absence of structural abnormalities predicts with near certainty the presence of autoantibodies against 60kD and 52kD SSA/Ro in the mother regardless of her health status. Previous studies have emphasized these autoantibodies as key mediators of tissue injury. The aim of this study was to focus on the anti-Ro52 response to determine whether these autoantibodies originate from progenitors that are inherently self-reactive or from B-cells that acquire self-reactivity during an immune response. We traced the evolution of two anti-Ro52 autoantibodies isolated from circulating IgG1-switched B-cells from an asymptomatic mother of a child with third degree congenital heart block. The autoantibodies were expressed as their immune form and as pre-immune ancestors by reverting somatic mutations to germline sequence. The reactivity of pre-immune and immune antibodies for Ro52, Ro60, La and DNA was measured. Both anti-Ro52 autoantibodies exhibited a low frequency of somatic mutations (3-4%) and utilised the same heavy and light chain genes but represented distinct clones based on differing complementarity determining region sequences. Pre- and post-immune antibodies showed specific binding to Ro52 with no measurable reactivity for other autoantigens. Ro52 binding was higher for immune antibodies compared to pre-immune counterparts demonstrating that autoreactivity was enhanced by affinity maturation. These data indicate that Ro52 reactivity is an intrinsic property of the germline antibody repertoire in a mother with a pathogenic antibody defined by cardiac injury in her offspring, and implies defects in both central and peripheral tolerance mechanisms.

Induction of neutralizing antibodies in rhesus macaques using V3 mimotope peptides.

RV144 vaccinees with low HIV-1 Envelope-specific IgA antibodies (Abs) also had Abs directed to the hypervariable region 3 (V3) that inversely correlated with infection risk. Thus, anti-V3 HIV-1 Abs may contribute to protection from HIV-1 infection. The V3 region contains two dominant clusters of epitopes; one is preferentially recognized by mAbs encoded by VH5-51 and VL lambda genes, while the second one is recognized by mAbs encoded by other VH genes. We designed a study in rhesus macaques to induce anti-V3 Abs specific to each of these two dominant clusters of V3 epitopes to test whether the usage of the VH5-51 gene results in different characteristics of antibodies. The two C4-V3 immunogens used for immunization were each comprised of a fusion of the C4 peptide containing the T cell epitope and a V3 mimotope peptide mimicking the V3 epitope. The C4-447 peptide was designed to target B cells with several VH1-VH4 genes, the C4-VH5-51 peptide was designed to specifically target B cells with the VH5-51 gene. Six animals in two groups were immunized five times with these two immunogens, and screening of 10 sequential plasma samples post immunization demonstrated that C4-447 induced higher titers of plasma anti-V3 Abs and significantly more potent neutralizing activities against tier 1 and some tier 2 pseudoviruses than C4-VH5-51. Levels of anti-V3 Abs in buccal secretions were significantly higher in sequential samples derived from C4-447- than from C4-VH5-51-immunized animals. The titers of anti-V3 Abs in plasma strongly correlated with their levels in mucosal secretions. The results show that high titers of vaccine-induced anti-V3 Abs in plasma determine the potency and breadth of neutralization, as well as the rate of transduction of Abs to mucosal tissues, where they can play a role in preventing HIV-1 infection.

A broad range of mutations in HIV-1 neutralizing human monoclonal antibodies specific for V2, V3, and the CD4 binding site.

The HIV vaccine-induced neutralizing antibodies (Abs) display low rates of mutation in their variable regions. To determine the range of neutralization mediated by similar human monoclonal Abs (mAbs) but derived from unselected chronically HIV-1 infected subjects, we tested a panel of 66 mAbs specific to V3, CD4 binding site (CD4bs) and V2 regions. The mAbs were tested against 41 pseudoviruses, including 15 tier 1 and 26 tier 2, 3 viruses, showing that the neutralization potency and breadth of anti-V3 mAbs were significantly higher than those of the anti-CD4bs and anti-V2 mAbs, and only anti-V3 mAbs were able to neutralize some tier 2, 3 viruses. The percentage of mutations in the variable regions of the heavy (VH) and light (VL) chains varied broadly in a range from 2% to 18% and correlated moderately with the neutralization breadth of tier 2, 3 viruses. There was no correlation with neutralization of tier 1 viruses as some mAbs with low and high percentages of mutations neutralized the same number of viruses. The electrostatic interactions between anti-V3 mAbs and the charged V3 region may contribute to their neutralization because the isoelectric points of the VH CDR3 of 48 anti-V3 mAbs were inversely correlated with the neutralization breadth of tier 2, 3 viruses. The results demonstrate that infection-induced antibodies to CD4bs, V3 and V2 regions can mediate cross-clade neutralization despite low levels of mutations which can be achieved by HIV-1 vaccine-induced antibodies.

Cross-neutralizing activity of human anti-V3 monoclonal antibodies derived from non-B clade HIV-1 infected individuals.

One approach to the development of an HIV vaccine is to design a protein template which can present gp120 epitopes inducing cross-neutralizing antibodies. To select a V3 sequence for immunogen design, we compared the neutralizing activities of 18 anti-V3 monoclonal antibodies (mAbs) derived from Cameroonian and Indian individuals infected with clade AG and C, respectively. It was found that V3 mAbs from the Cameroonian patients were significantly more cross-neutralizing than those from India. Interestingly, superior neutralizing activity of Cameroonian mAbs was also observed among the nine VH5-51/VL lambda genes encoding V3 mAbs which mediate a similar mode of recognition. This correlated with higher relative binding affinity to a variety of gp120s and increased mutation rates in V3 mAbs from Cameroon. These results suggest that clade C V3 is probably weakly immunogenic and that the V3 sequence of CRF02_AG viruses can serve as a plausible template for vaccine immunogen design.

Different pattern of immunoglobulin gene usage by HIV-1 compared to non-HIV-1 antibodies derived from the same infected subject.

A biased usage of immunoglobulin (Ig) genes is observed in human anti-HIV-1 monoclonal antibodies (mAbs) resulting probably from compensation to reduced usage of the VH3 family genes, while the other alternative suggests that this bias usage is due to antigen requirements. If the antigen structure is responsible for the preferential usage of particular Ig genes, it may have certain implications for HIV vaccine development by the targeting of particular Ig gene-encoded B cell receptors to induce neutralizing anti-HIV-1 antibodies. To address this issue, we have produced HIV-1 specific and non-HIV-1 mAbs from an infected individual and analyzed the Ig gene usage. Green-fluorescence labeled virus-like particles (VLP) expressing HIV-1 envelope (Env) proteins of JRFL and BaL and control VLPs (without Env) were used to select single B cells for the production of 68 recombinant mAbs. Ten of these mAbs were HIV-1 Env specific with neutralizing activity against V3 and the CD4 binding site, as well as non-neutralizing mAbs to gp41. The remaining 58 mAbs were non-HIV-1 Env mAbs with undefined specificities. Analysis revealed that biased usage of Ig genes was restricted only to anti-HIV-1 but not to non-HIV-1 mAbs. The VH1 family genes were dominantly used, followed by VH3, VH4, and VH5 among anti-HIV-1 mAbs, while non-HIV-1 specific mAbs preferentially used VH3 family genes, followed by VH4, VH1 and VH5 families in a pattern identical to Abs derived from healthy individuals. This observation suggests that the biased usage of Ig genes by anti-HIV-1 mAbs is driven by structural requirements of the virus antigens rather than by compensation to any depletion of VH3 B cells due to autoreactive mechanisms, according to the gp120 superantigen hypothesis.

Functional and immunochemical cross-reactivity of V2-specific monoclonal antibodies from HIV-1-infected individuals.

The recent analysis of the first successful RV144 vaccine trial revealed that a high titer of plasma anti-V2 antibodies (Abs) correlated with a decreased risk of HIV-1 infection in vaccine recipients. To understand the mechanism of immune correlates, we studied seven anti-V2 monoclonal Abs (mAbs) developed from HIV-1 infected individuals. The V2 mAbs target conserved epitopes, including the binding site for α4ß7 integrin, and are broadly cross-reactive with various gp120 proteins. Preferential usage of the VH1-69 gene by V2 mAbs may depend on selection by the same antigenic structure. Six of seven V2 mAbs weakly neutralized four to eight of the 41 pseudoviruses tested and resistance to neutralization was correlated with longer V2 domains. The data suggest the presence of shared, conserved structural elements in the V2 loop, and these can be used in the design of vaccine immunogens inducing broadly reactive Abs with anti-viral activities.

Clonal analysis of human anti-V3 monoclonal antibodies selected by a V3 tetramer.

The production of human monoclonal antibodies (mAbs) has been improved recently using the single B cell and PCR technology. A number of new anti-HIV-1 mAbs directed to various epitopes were produced by selecting single B cells from HIV positive individuals using the HIV-1 envelope (Env) proteins, and we tested whether the peptide can select B cells specific to a particular Env epitope. Using the fluorescently-labeled peptide tetramer representative of the V3 loop of HIV-1 Env gp120 for staining the B cells derived from one HIV-1 infected donor, four clonal human mAbs were produced with specificity to the V3 region. The clonality of the four V3 mAbs was based on the usage of the same immunoglobulin genes and almost identical sequence of CDRs. The amino acid changes were present only in the framework and, possibly, they could be related to the differences observed in the relative affinity binding of these four mAbs to V3 antigen. One representative V3 mAb displayed very potent neutralizing activity to one of two viruses tested. This study shows the feasibility of utilizing a peptide tetramer to select epitope-specific B cells and produce mAbs.

Human anti-V3 HIV-1 monoclonal antibodies encoded by the VH5-51/VL lambda genes define a conserved antigenic structure.

Preferential usage of immunoglobulin (Ig) genes that encode antibodies (Abs) against various pathogens is rarely observed and the nature of their dominance is unclear in the context of stochastic recombination of Ig genes. The hypothesis that restricted usage of Ig genes predetermines the antibody specificity was tested in this study of 18 human anti-V3 monoclonal Abs (mAbs) generated from unrelated individuals infected with various subtypes of HIV-1, all of which preferentially used pairing of the VH5-51 and VL lambda genes. Crystallographic analysis of five VH5-51/VL lambda-encoded Fabs complexed with various V3 peptides revealed a common three dimensional (3D) shape of the antigen-binding sites primarily determined by the four complementarity determining regions (CDR) for the heavy (H) and light (L) chains: specifically, the H1, H2, L1 and L2 domains. The CDR H3 domain did not contribute to the shape of the binding pocket, as it had different lengths, sequences and conformations for each mAb. The same shape of the binding site was further confirmed by the identical backbone conformation exhibited by V3 peptides in complex with Fabs which fully adapted to the binding pocket and the same key contact residues, mainly germline-encoded in the heavy and light chains of five Fabs. Finally, the VH5-51 anti-V3 mAbs recognized an epitope with an identical 3D structure which is mimicked by a single mimotope recognized by the majority of VH5-51-derived mAbs but not by other V3 mAbs. These data suggest that the identification of preferentially used Ig genes by neutralizing mAbs may define conserved epitopes in the diverse virus envelopes. This will be useful information for designing vaccine immunogen inducing cross-neutralizing Abs.

Downregulation of SGK1 by nucleotides in renal tubular epithelial cells.

This study determined whether nucleotides that bind to purinergic receptors (P2R) regulate the expression or function of serum- and glucocorticoid-inducible kinase-1 (SGK1) in mouse renal inner medullar collecting duct cells (mIMCD-3). The SGK1 protein was detected by Western blotting. A significant reduction of cytosolic SGK1 expression was observed in the cells pretreated with P2R agonist adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), and the reduction could be reversed by P2R antagonists. This reduction was also observed in cells that were pretreated with agonists for P2R subtypes. Using ELISA, we observed a reduced SGK1 kinase activity in ATPgammaS-pretreated cells. This effect was reversed by P2R antagonists. Furthermore, an increase of SGK1 kinase activity in aldosterone-pretreated cells was suppressed by ATPgammaS. These studies demonstrate for the first time that SGK1 can be downregulated by nucleotides in renal collecting duct epithelial cells, likely via the activation of P2R, and suggest that activation of renal purinergic signaling regulates a SGK1-dependent pathway that is known to modulate ion transport in the renal collecting duct.

Apical P2XR contribute to [Ca2+]i signaling and Isc in mouse renal MCD.

We examined P2X receptor expression and distribution in the mouse collecting duct (CD) and their functional role in Ca(2+) signaling. Both P2X(1) and P2X(4) were detected by RT-PCR and Western blot. Immunohistochemistry demonstrated apical P2X(1) and P2X(4) immunoreactivity in principal cells in the outer medullary CD (OMCD) and inner medullary CD (IMCD). Luminal ATP induced an increase in Ca(2+) signaling in native medullary CD (MCD) as measured by fluorescence imaging. ATP also induced an increase in Ca(2+) signaling in MCD cells grown in primary culture but not in the presence of P2XR antagonist PPNDS. Short circuit current (I(sc)) measurement with mouse IMCD cells showed that P2XR agonist BzATP induced a larger I(sc) than did P2YR agonist UTP in the apical membrane. Our data reveal for the first time that P2X(1) and P2X(4) are cell-specific with prominent immunoreactivity in the apical area of MCD cells. The finding that P2XR blockade inhibits ATP-induced Ca(2+) signaling suggests that activation of P2XR is a key step in Ca(2+)-dependent purinergic signaling. The result that activation of P2XR produces large I(sc) indicates the necessity of P2XR in renal CD ion transport.

Nitric oxide upregulation of caspase-8 mRNA expression in lung endothelial cells: role of JAK2/STAT-1 signaling.

We recently reported that nitric oxide (NO) modulates expression of multiple genes associated with apoptotic pathways, including expression of caspase-8. The objective of the present study is to determine whether the NO-induced expression of the caspase-8 gene is regulated via signal transducers and activators of transcription-1 (STAT-1) signaling. The confluent monolayers of pulmonary artery endothelial cells (PAEC) were incubated with or without (control) 1 mM NOC-18, a NO donor, at 37 degrees C for 0-24 h. In some experiments PAEC were pretreated with a Janus kinase (JAK-2) inhibitor, AG490 (20 microM). Exposure of PAEC to NO-increased relative levels of caspase-8 mRNA as determined using quantitative real time PCR. Relative levels of phosphorylated STAT-1 at Serine (Ser)-727, but not total STAT-1 expression in NO-exposed cells, were upregulated significantly compared to control cells. AG490 attenuated NO-induced phosphorylation of STAT-1 at Ser 727 and expression of caspase-8 mRNA, suggesting JAK2 plays a role in the induction of caspase-8 mRNA. The promoter of caspase-8 has four gamma-activated sequence (GAS) and two interferon-stimulated response element (ISRE) transcription factor-binding sites. NO enhanced the STAT-1 binding activity to GAS/ISRE. Suppression of STAT-1 expression attenuated NO-induced elevation of caspase-8 mRNA. These studies demonstrate that a NO-dependent increase in caspase-8 mRNA levels is associated with phosphorylation of STAT-1 at Ser-727 and STAT1 binding to the caspase-8 promoter in cultured PAEC.

[Studies on the protective effect of the mutant of Sj23 DNA vaccine against schistosomiasis].

OBJECTIVE: To investigate the protective immunity of the vaccine against schistosomiasis, a mutant of Mr 23 000 membrane protein DNA (Sj23DNA) without the homologous sequence of ME491. METHODS: The mutant of Sj23 DNA with no homologous sequence of ME491 on the cell membrane of human melanoma was obtained by overlap PCR. The mutant was transfected into human embryonic kidney cells of the line HEK293. Indirect fluorescent antibody test (IFAT) was used to detect the expressed protein. Expression of the mutant of Sj23DNA in muscular cells of mice was conducted through vaccinating the mouse with 100 microg purified plasmids by injecting them into the quadriceps muscle of thigh. Four weeks after the immunization, the quadriceps muscles were taken and cryostat sections were prepared for detecting the expression by IFAT. Forty BALB/c mice were randomly divided into four groups and injected with the mutant of pcDNA3-Sj23 plasmid DNA, pcDNA3-Sj23 plasmid DNA, pcDNA3 blank plasmid (100 microg per mouse) and sterile saline (30 microl per mouse) respectively. Four weeks after the immunization, mice were challenged with cercariae (40+/-2 cercariae per mouse) by abdominal skin penetration. Mice were then killed 6 weeks later, perfusion and squash methods were carried out to collect the adult worms and the number of eggs per gram of liver tissue was calculated. Worm and egg reduction rates were used to evaluate the protective immunity. RESULTS: Specific fluorescence was demonstrated in muscular cells of mice vaccinated with the mutant of pcDNA3-Sj23. The worm reduction rate and egg reduction rate were 40.3% and 42.8% respectively in the mutant of pcDNA3-Sj23 group, which were higher than those in the pcDNA3-Sj23 plasmid group (33.1% and 28.9% respectively). The difference between these two groups was significant (P<0.05). CONCLUSION: The modified Sj23DNA without the homologous sequence of ME491 induces higher protection against Schistosoma japonicum infection in mice than that of Sj23DNA.