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The vacuolar H + -ATPase (V-ATPase) is an ATP-dependent proton pump that functions to control the pH of intracellular compartments as well as to transport protons across the plasma membrane of various cell types, including cancer cells. We have previously shown that selective inhibition of plasma membrane V-ATPases in breast tumor cells inhibits the invasion of these cells in vitro . We have now developed a nanobody directed against an extracellular epitope of the mouse V-ATPase c subunit. We show that treatment of 4T1-12B mouse breast cancer cells with this nanobody inhibits V-ATPase-dependent acidification of the media and invasion of these cells in vitro . We further find that injection of this nanobody into mice implanted with 4T1-12B cells orthotopically in the mammary fat pad inhibits metastasis of tumor cells to lung. These results suggest that plasma membrane V-ATPases represent a novel therapeutic target to limit breast cancer metastasis.

Despite accumulating evidence linking defective lysosome function with autoimmune diseases, how the catabolic machinery is regulated to maintain immune homeostasis remains unknown. Late endosomal/lysosomal adaptor, MAPK and mTOR activator 5 (Lamtor5) is a subunit of the Ragulator mediating mechanistic target of rapamycin complex 1 (mTORC1) activation in response to amino acids, but its action mode and physiological role are still unclear. Here it is demonstrated that Lamtor5 level is markedly decreased in peripheral blood mononuclear cells (PBMCs) of patients with systemic lupus erythematosus (SLE). In parallel, the mice with myeloid Lamtor5 ablation developed SLE-like manifestation. Impaired lysosomal function and aberrant activation of mTORC1 are evidenced in Lamtor5 deficient macrophages and PBMCs of SLE patients, accompanied by blunted autolysosomal pathway and undesirable inflammatory responses. Mechanistically, it is shown that Lamtor5 is physically associated with ATP6V1A, an essential subunit of vacuolar H + -ATPase (v-ATPase), and promoted the V0/V1 holoenzyme assembly to facilitate lysosome acidification. The binding of Lamtor5 to v-ATPase affected the lysosomal tethering of Rag GTPase and weakened its interaction with mTORC1 for activation. Overall, Lamtor5 is identified as a critical factor for immune homeostasis by intergrading v-ATPase activity, lysosome function, and mTOR pathway. The findings provide a potential therapeutic target for SLE and/or other autoimmune diseases., (© 2024 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

Abnormal ATPase H+ Transporting Accessory Protein 1 ( ATP6AP1 ) expression may promote carcinogenesis. We investigated the association of ATP6AP1 with breast cancer (BC) and COVID-19. The Oncomine, Gene Expression Profiling Interactive Analysis, Human Protein Atlas and Kaplan-Meier plotter databases were used to evaluate the expression and prognostic value of ATP6AP1 in BC. ATP6AP1 was upregulated in BC tissues, and higher ATP6AP1 expression was associated with poorer outcomes. Data from the Tumor Immune Estimation Resource, Tumor-Immune System Interaction Database and Kaplan-Meier plotter indicated that ATP6AP1 expression correlated with immune infiltration, and that its prognostic effects in BC depended on tumor-infiltrating immune cell subtype levels. Multiple databases were used to evaluate the association of ATP6AP1 with clinicopathological factors, assess the mutation and methylation of ATP6AP1 , and analyze gene co-expression and enrichment. The ATP6AP1 promoter was hypomethylated in BC tissues and differentially methylated between different disease stages and subtypes. Data from the Gene Expression Omnibus indicated that ATP6AP1 levels in certain cell types were reduced after SARS-CoV-2 infections. Ultimately, higher ATP6AP1 expression was associated with a poorer prognosis and with higher or lower infiltration of particular immune cells in BC. BC patients may be particularly susceptible to SARS-CoV-2 infections, which may alter their prognoses.

Vitamin D and beta-glucans are both immunostimulants. Vitamin D exerts its beneficial effects on many components of the immune system. In macrophages, the hormone modulates both phagocytic activity and cytokine production; therefore, it plays an important role in mediating the innate immune response to infection. The immunomodulatory properties of beta-glucans are attributed to the ability of these fungal cell wall polysaccharides to bind to different receptors expressed on the cell surface of phagocytic and cytotoxic innate immune cells, including monocytes and macrophages. The intracellular signaling pathways activated by beta-glucans lead to enhanced phagocytosis and cytokine response. In this study we investigated the possible potentiation of immunomodulatory properties of the combined treatment with vitamin D and beta-glucans. The effects of 100 nM 1,25-dihydroxyvitamin D3 or 100 µg/mL beta-glucans were evaluated in human macrophages in terms of cytokine production, intracellular vesicle acidification and changes in energy metabolism, three hallmarks of macrophage antimicrobial activation. We found that all the analyzed parameters were enhanced by the co-treatment compared to the response to single molecules. The results of this study support the validity of a novel therapeutic approach that could boost the immune response, taking advantage of the synergy between two natural compounds.

Despite recent progress in hepatitis treatment, there have been no significant advances in the development of liver cancer vaccines in recent years. In this study, we investigated the regulatory effect and potential mechanism of hepatocyte growth factor receptor (MET, also known as HGFR) on tumor vaccinations for liver cancer in mice. Herein, we demonstrate that MET expression is significantly associated with the immunogenicity of liver cancer in mice and humans, and that MET depletion dramatically enhances the protective efficacy of chemotherapy-based anti-liver cancer vaccination. Mechanistically, MET repressed liver cancer immunogenicity independent of the traditional PI3K-AKT cascade, and MET interacted with vacuolar ATP synthase (V-ATPase) and mediated the activation of mammalian target of rapamycin (MTOR), thus suppressing liver cancer immunogenicity. The efficacy of chemotherapy-based liver cancer vaccination was markedly enhanced by targeting the MET-V-ATPase-MTOR axis, highlighting a translational strategy for identifying MET-associated drug candidates for cancer prevention.

Alternative (M2)-polarized macrophages possess high capacities to produce specialized proresolving mediators (SPM; i.e., resolvins, protectins, and maresins) that play key roles in resolution of inflammation and tissue regeneration. Vacuolar (H + )-ATPase (V-ATPase) is fundamental in inflammatory cytokine trafficking and secretion and was implicated in macrophage polarization toward the M2 phenotype, but its role in SPM production and lipid mediator biosynthesis in general is elusive. In this study, we show that V-ATPase activity is required for the induction of SPM-biosynthetic pathways in human M2-like monocyte-derived macrophages (MDM) and consequently for resolution of inflammation. Blockade of V-ATPase by archazolid during IL-4-induced human M2 polarization abrogated 15-lipoxygenase-1 expression and prevented the related biosynthesis of SPM in response to pathogenic Escherichia coli , assessed by targeted liquid chromatography-tandem mass spectrometry-based metabololipidomics. In classically activated proinflammatory M1-like MDM, however, the biosynthetic machinery for lipid mediator formation was independent of V-ATPase activity. Targeting V-ATPase in M2 influenced neither IL-4-triggered JAK/STAT6 nor the mTOR complex 1 signaling but strongly suppressed the ERK-1/2 pathway. Accordingly, the ERK-1/2 pathway contributes to 15-lipoxygenase-1 expression and SPM formation in M2-like MDM. Targeting V-ATPase in vivo delayed resolution of zymosan-induced murine peritonitis accompanied by decreased SPM levels without affecting proinflammatory leukotrienes or PGs. Together, our data propose that V-ATPase regulates 15-lipoxygenase-1 expression and consequent SPM biosynthesis involving ERK-1/2 during M2 polarization, implying a crucial role for V-ATPase in the resolution of inflammation., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Objective: Concomitant presence of renal tubular acidosis (RTA) and autoimmune diseases is indicative of the potential role of immune factors in the pathogenesis of RTA. Our study aimed to detect the serum antibodies to renal tubular epithelial cells in RTA patients., Methods: We enrolled 11 RTA patients, eight primary Sjögren's syndrome (pSS) patients and eight healthy controls (HC). Serum biochemical test, urinary regular test, and 24 hours urinary protein quantification were measured using a fully automated analyzer. Immunofluorescence assay was performed to detect the antibodies to subunit B1 and subunit B2 of v-H+-ATPases (adenosine triphosphatases) in the serum of the participants., Results: Clinically, RTA patients showed hyperchloremia, acidosis and paradoxical alkaline urine. We detected the serum antibodies to renal tubular epithelial cells and there were 6/11 positive in RTA patients, much higher than that in the pSS group (0/8) and the HC group (0/8). Subsequently, we demonstrated that in normal renal tissue, the B1 subunit of v-H+-ATPase specifically expressed in intercalated cells, while the B2 subunit continuously expressed along the lumen of renal tubular epithelial cells. Moreover, the antibody to subunit B1/B2 of v-H+-ATPase was positive in the sera of 6 RTA patients (54%), while it was negative in both the pSS and HC group., Conclusions: We detected the presence of serum autoantibodies to subunit B1 and subunit B2 of v-H + -ATPase in RTA patients. Our findings may provide novel mechanism insights into the pathogenesis of RTA and the potential diagnostic utility of antibodies to v-H + -ATPase in the classification of RTA., (© 2019 Asia Pacific League of Associations for Rheumatology and John Wiley & Sons Australia, Ltd.)

Osteoimmunological studies have revealed that T cells exert a powerful impact on the formation and activity of osteoclasts and bone remodeling. Evidence demonstrates that immature dendritic cells (iDCs) are more efficient transdifferentiating into osteoclasts (OCs) than monocytes. However, whether Vγ9Vδ2 T (γδ T) cells stimulate or inhibit iDC transdifferentiation into OCs has never been reported. The aim of the present study was to investigate the effects of γδ T cells on this transdifferentiation process. γδ T cells and iDCs were isolated from the peripheral blood of healthy volunteers separately and were co‑cultured with Transwelll inserts, with γδ T cells in the upper chamber and iDCs in the lower chamber. IDCs were treated with macrophage‑colony stimulating factor and receptor activator of nuclear factor‑κB (RANK) ligand. Tartrate resistant acid phosphatase (TRAP) assay and dentine resorption assay were performed to detect OC formation and their resorption capacity, respectively. The mRNA expression of OCs was examined using a microarray and real time‑quantitative polymerase chain reaction to trace the changes during iDC transdifferentiation into OCs. The results demonstrated that γδ T cells significantly inhibited the generation of the TRAP‑positive OCs from iDCs and their resorption capacity. The microarray analysis identified decreased expression level of Fos proto‑oncogene AP‑1 transcription factor subunit (c‑Fos), ATPase H+ transporting V0 subunit d (ATP6V0D2) and cathepsin K when iDCs were co‑cultured with γδ T cells. These genes are associated with OC differentiation, indicating that γδ T cells suppressed iDCs osteoclastogenesis by downregulation of the RANK/c‑Fos/ATP6V0D2 signaling pathway. The present findings provide novel insights into the interactions between human γδ T cells and iDCs, and demonstrate that γδ T cells are capable of inhibiting OC formation and their activity via downregulation of genes associated with OC differentiation.

The urinary tract is usually culture negative despite its close proximity to microbial flora. The precise mechanism by which the kidneys and urinary tract defends against infection is not well understood. The initial kidney cells to encounter ascending pathogens are the collecting tubule cells that consist of principal cells (PCs) that express aquaporin 2 (AQP2) and intercalated cells (ICs) that express vacuolar H + -ATPase (V-ATPase, B1 subunit). We have previously shown that ICs are involved with the human renal innate immune defense. Here we generated two reporter mice, VATPase B1-cre + tdT + mice to fluorescently label ICs and AQP2-cre + tdT + mice to fluorescently label PCs, and then performed flow sorting to enrich PCs and ICs for analysis. Isolated ICs and PCs along with proximal tubular cells were used to measure antimicrobial peptide (AMP) mRNA expression. ICs and PCs were significantly enriched for AMPs. Isolated ICs responded to uropathogenic Escherichia coli (UPEC) challenge in vitro and had higher RNase4 gene expression than control while both ICs and PCs responded to UPEC challenge in vivo by upregulating Defb1 mRNA expression. To our knowledge, this is the first report of isolating murine collecting tubule cells and performing targeted analysis for multiple classes of AMPs.

A monoclonal antibody (mAb) G2 possesses an unusual characteristic of reacting with at least three proteins (ATP6V1C1, SEPT3, and C6H10orf76) other than its original antigen, chicken prion protein (ChPrP). The epitopes on ChPrP and ATP6V1C1 have been identified previously. In this study, we identified the epitope in the third protein, SEPT3. Interestingly, there was no amino acid sequence similarity among the epitopes on the three proteins. These epitopes had high binding affinities to G2 (K D  = ∼10 -7 M for monovalent binding and K D  = ∼10 -9 M for divalent binding), as determined using a SPR biosensor. This is the first report on a three-in-one mAb recognizing completely different epitope sequences with high affinity. Additionally, competitive ELISA indicated that the binding sites on G2, specific for the three different epitopes, overlapped, suggesting that the antigen-binding site may be flexible in the free form and capable of adapting to at least three different conformations to enable interactions with three different antigens., (© 2017 The Protein Society.)

Macrophages play crucial roles in host defence and tissue homoeostasis, processes in which both environmental stimuli and intracellularly generated metabolites influence activation of macrophages. Activated macrophages are classified into M1 and M2 macrophages. It remains unclear how intracellular nutrition sufficiency, especially for amino acid, influences on macrophage activation. Here we show that a lysosomal adaptor protein Lamtor1, which forms an amino-acid sensing complex with lysosomal vacuolar-type H + -ATPase (v-ATPase), and is the scaffold for amino acid-activated mTORC1 (mechanistic target of rapamycin complex 1), is critically required for M2 polarization. Lamtor1 deficiency, amino-acid starvation, or inhibition of v-ATPase and mTOR result in defective M2 polarization and enhanced M1 polarization. Furthermore, we identified liver X receptor (LXR) as the downstream target of Lamtor1 and mTORC1. Production of 25-hydroxycholesterol is dependent on Lamtor1 and mTORC1. Our findings demonstrate that Lamtor1 plays an essential role in M2 polarization, coupling immunity and metabolism.

Several receptor proteins of Cry toxin have been previously identified, including cadherin-like, aminopeptidase N, and alkaline phosphatase. In the present work, a novel binding protein, V-ATPase subunit A (HpVAA), was identified in Holotricia parallela larvae and characterized. We performed reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends technology to obtain the cDNA of the full-length hpvaa. Sequencing analysis showed that the open reading frame of hpvaa (GenBank accession No. KU497557) is 1845 bp long, encoding 614 amino acid residues. The predicted molecular weight and isoelectric point of HpVAA were 67.85 kDa and 4.9, respectively. The HpVAA protein, which includes two putative conserved domains, ATP-synt_ab_N and ATP-synt_ab_C, and a Walker A (GAFGCGKT) motif and a Walker B (SMMAD) motif, possesses the same structural characteristics as V-ATPase subunit A from other insects. The protein was successfully expressed in Escherichia coli, and a ligand blot assay showed binding of the protein with Cry8Ea3 toxin. Transcriptional analysis of hpvaa in different tissues of H. parallela larvae was performed by qRT-PCR, which showed that the relative expression of hpvaa in the Malpighian tubules is higher than that in other tissues.

Introduction: Previously, we demonstrated the importance of T-cell immune response cDNA 7 (TIRC7) in acute immune thrombocytopenia (ITP). As the downstream molecule of TIRC7, cytotoxic T lymphocyte antigen-4 (CTLA-4) has been verified its negative regulation of acute ITP. This study aimed to investigate the exact role of CTLA-4 and its relationship with TIRC7 in acute ITP., Patients and Methods: 37 patients with acute ITP were enrolled and received dexamethasone (40mg/day) for 4 consecutive days. Patients who had platelet counts more than 50×10(9)/L or less were defined as responders or non-responders after treatment. The plasma, protein and mRNA levels of CTLA-4 and TIRC7 were monitored by ELISA, western blot and q-PCR, respectively., Results: After high-dose dexamethasone therapy, CTLA-4 levels were significantly elevated not only in acute ITP patients (P<0.001; P<0.0001) but also in acute ITP responders (P<0.0001; P<0.0001). The levels of CTLA-4 were negatively correlated with the levels of TIRC7 before and after treatment; IFN-γ (Th1), IL-17 (Th17) and IL-22 (Th22) levels were all elevated, which were decreased after treatment not only in patients with acute ITP (P<0.01) but also in acute ITP responders (P<0.01)., Conclusions: CTLA-4 level might reflect treatment efficacy and it might be associated with the pathogenesis of acute ITP., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Inflammatory cytokines produced at the early stages of malaria infection contribute to shaping protective immunity and pathophysiology. To gain mechanistic insight into these processes, it is important to understand the cellular origin of cytokines because both cytokine input and cytokine-producing cells play key roles. Here, we determined cytokine responses by monocytes, macrophages, and dendritic cells (DCs) to purified Plasmodium falciparum and Plasmodium berghei ANKA, and by spleen macrophages and DCs from Plasmodium yoelii 17NXL-infected and P. berghei ANKA-infected mice. The results demonstrate that monocytes and macrophages do not produce inflammatory cytokines to malaria parasites and that DCs are the primary source early in infection, and DC subsets differentially produce cytokines. Importantly, blocking of phagosomal acidification by inhibiting vacuolar-type H(+)-ATPase enabled macrophages to elicit cytokine responses. Because cytokine responses to malaria parasites are mediated primarily through endosomal Toll-like receptors, our data indicate that the inability of macrophages to produce cytokines is due to the phagosomal acidification that disrupts endosomal ligand-receptor engagement. Macrophages efficiently produced cytokines to LPS upon simultaneously internalizing parasites and to heat-killed Escherichia coli, demonstrating that phagosomal acidification affects endosomal receptor-mediated, but not cell surface receptor-mediated, recognition of Toll-like receptor agonists. Enabling monocytes/macrophages to elicit immune responses to parasites by blocking endosomal acidification can be a novel strategy for the effective development of protective immunity to malaria. The results have important implications for enhancing the efficacy of a whole parasite-based malaria vaccine and for designing strategies for the development of protective immunity to pathogens that induce immune responses primarily through endosomal receptors., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Neutrophils kill microorganisms by inducing exocytosis of granules with antibacterial properties. Four isoforms of the "a" subunit of V-ATPase-a1V, a2V, a3V, and a4V-have been identified. a2V is expressed in white blood cells, that is, on the surface of monocytes or activated lymphocytes. Neutrophil associated-a2V was found on membranes of primary (azurophilic) granules and less often on secondary (specific) granules, tertiary (gelatinase granules), and secretory vesicles. However, it was not found on the surface of resting neutrophils. Following stimulation of neutrophils, primary granules containing a2V as well as CD63 translocated to the surface of the cell because of exocytosis. a2V was also found on the cell surface when the neutrophils were incubated in ammonium chloride buffer (pH 7.4) a weak base. The intracellular pH (cytosol) became alkaline within 5 min after stimulation, and the pH increased from 7.2 to 7.8; this pH change correlated with intragranular acidification of the neutrophil granules. Upon translocation and exocytosis, a2V on the membrane of primary granules remained on the cell surface, but myeloperoxidase was secreted. V-ATPase may have a role in the fusion of the granule membrane with the cell surface membrane before exocytosis. These findings suggest that the granule-associated a2V isoform has a role in maintaining a pH gradient within the cell between the cytosol and granules in neutrophils and also in fusion between the surface and the granules before exocytosis. Because a2V is not found on the surface of resting neutrophils, surface a2V may be useful as a biomarker for activated neutrophils., (© Society for Leukocyte Biology.)

Breast cancer represents a significant health problem because of its high prevalence. Tests like mammography, which are used abundantly for the detection of breast cancer, suffer from serious limitations. Mammography correctly detects malignancy about 80-90% of the times, failing in places when (1) the tumor is small at early stage, (2) breast tissue is dense or (3) in women of less than 40 years. Serum-based detection of biomarkers involves risk of disease transfer, along with other concerns. These techniques compromise in the early detection of breast cancer. Early detection of breast cancer is a crucial factor to enhance the survival rate of patient. Development of regular screening tests for early diagnosis of breast cancer is a challenge. This review highlights the design of a handy and household biosensor device aimed for self-screening and early diagnosis of breast cancer. The design makes use of salivary autoantibodies for specificity to develop a noninvasive procedure, breast cancer specific biomarkers for precision for the development of device, and biosensor technology for sensitivity to screen the early cases of breast cancer more efficiently., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The augmentation of high-titer antibodies to ATP6S1 is associated with favorable clinical outcomes in patients who received vaccination with autologous, irradiated tumor cells engineered to secrete GM-CSF and allogeneic bone marrow transplantation. Cellular immune responses to ATP6S1 are unknown. To define its role as an immune target, examination of cellular responses to ATP6S1 and immunity related to current therapies such as checkpoint blockade is needed. We used an overlapping peptide library representing the full-length ATP6S1 protein to screen for cellular responses from the peripheral blood of patients with stage III and IV melanoma. Reactive peptide pools were used to determine the individual peptide activity and epitopes. Recombinant ATP6S1 protein was used in an ELISA to assess potential correlation with humoral immune responses and changes in immunity related to CTLA-4 blockade with ipilimumab in these patients. We observed a broad array of CD4(+) and CD8(+) cellular responses against ATP6S1, including the identification of several MHC class I and II ATP6S1 epitopes. The generation of specific CD4(+) and cytotoxic T cells revealed potent functional capability elicited by ipilimumab treatment in patients with metastatic melanoma, which revealed potent functional capability, including cytokine production, proliferation responsiveness to melanoma cell lines, and tumor-cell killing. Furthermore, the augmented humoral immune responses to ATP6S1 as a function of ipilimumab treatment were associated with beneficial clinical outcomes. These results support the continued development of ATP6S1 as a biomarker and therapeutic target., (©2014 American Association for Cancer Research.)

SLC15A4 is a lysosome-resident, proton-coupled amino-acid transporter that moves histidine and oligopeptides from inside the lysosome to the cytosol of eukaryotic cells. SLC15A4 is required for Toll-like receptor 7 (TLR7)- and TLR9-mediated type I interferon (IFN-I) productions in plasmacytoid dendritic cells (pDCs) and is involved in the pathogenesis of certain diseases including lupus-like autoimmunity. How SLC15A4 contributes to diseases is largely unknown. Here we have shown that B cell SLC15A4 was crucial for TLR7-triggered IFN-I and autoantibody productions in a mouse lupus model. SLC15A4 loss disturbed the endolysosomal pH regulation and probably the v-ATPase integrity, and these changes were associated with disruption of the mTOR pathway, leading to failure of the IFN regulatory factor 7 (IRF7)-IFN-I regulatory circuit. Importantly, SLC15A4's transporter activity was necessary for the TLR-triggered cytokine production. Our findings revealed that SLC15A4-mediated optimization of the endolysosomal state is integral to a TLR7-triggered, mTOR-dependent IRF7-IFN-I circuit that leads to autoantibody production., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background and Objective: Interactions between TIRC7 (a novel seven-transmembrane receptor on activated lymphocytes) and its ligand HLA-DR might be involved in the inflammatory process in multiple sclerosis (MS)., Methods: Methods comprised immunohistochemistry and microscopy on archival MS autopsies, proliferation-, cytokine-, and surface-staining assays using peripheral blood lymphocytes (PBLs) from MS patients and an in vitro model., Results: TIRC7 was expressed in brain-infiltrating lymphocytes and strongly correlated with disease activity in MS. TIRC7 expression was reduced in T cells and induced in B cells in PBLs obtained from MS patients. After ex vivo activation, T cell expression of TIRC7 was restored in patients with active MS disease. The interaction of TIRC7(+) T lymphocytes with cells expressing HLA-DR on their surface led to T cell proliferation and activation whereas an anti-TIRC7 mAb preventing interactions with its ligand inhibited proliferation and Th1 and Th17 cytokine expression in T cells obtained from MS patients and in myelin basic protein-specific T cell clone., Conclusion: Our findings suggest that TIRC7 is involved in inflammation in MS and anti-TIRC7 mAb can prevent immune activation via selective inhibition of Th1- and Th17-associated cytokine expression. This targeting approach may become a novel treatment option for MS., (© The Author(s) 2014.)

a2V is required for maturation of sperm. The decreased expression of a2V at the feto-maternal interphase causes poor pregnancy outcome. The present study examined the role of a2V in spermatogenesis and inflammatory network in the testis. A single dose of anti-a2V mouse IgG or mouse IgG isotype (3 μg/animal) was injected i.p. into male mice on alternate days for 10 days. Anti-a2V-treated males exhibit severe deficiencies of spermatogenesis, which is indicated by the presence of less numbers of postmeiotic cells. Sperm counts and sperm motility were reduced significantly in anti-a2V-treated males. The release of the cleaved a2NTD was significantly lower in anti-a2V-treated testes. The TMs were identified as M2-like macrophages, and this population and the expression of various cytokines/chemokines (Tgf-β, Il-6, Nos2, Tnf, Lif, Mcp1, Ccl5) were decreased significantly in anti-a2V-treated testis compared with control testis. Moreover, the cleaved a2NTD acts as a key mediator of TMs and significantly up-regulates the secretion of testicular cytokines/chemokines, which are associated with normal spermatogenesis. When these anti-a2V-treated males were used for mating with normal females, the number of implantation sites was decreased significantly in the females mated with anti-a2V-treated males than the females mated with control males. These observations suggest that a2V plays a crucial role in spermatogenesis by regulating testicular immune responses, and its inhibition in males leads to poor pregnancy outcome in females., (© 2014 Society for Leukocyte Biology.)

Production of ROS and maintenance of an appropriate pH within the lumen of neutrophil and macrophage phagosomes are important for an effective immune response. Hv1 proton channels sustain ROS production at the plasma membrane, but their role in phagosomes is not known. Here, we tested whether Hv1 channels regulate the pH p and sustain phagosomal ROS production in neutrophils and macrophages. The presence of Hv1 channels on phagosomes of human neutrophils and mouse macrophages was confirmed by Western blot and immunostaining. Phagosomal ROS production, measured with OxyBurst-coupled targets, was reduced in neutrophils and macrophages isolated from Hv1-deficient mice. Ratiometric imaging of FITC-coupled targets showed that phagosomes acidified more slowly in Hv1-deficient macrophages and transiently alkalinized when the V-ATPase was inhibited. In WT neutrophils, 97% of phagosomes remained neutral 30 min after particle ingestion, whereas 37% of Hv1-deficient phagosomes were alkaline (pH>8.3) and 14% acidic (pH<6.3). The subpopulation of acidic phagosomes was eliminated by V-ATPase inhibition, whereas NOX inhibition caused a rapid acidification, independently of Hv1 expression. Finally, V-ATPase accumulation on phagosomes was inversely correlated to intraphagosomal ROS production in neutrophils. These data indicate that Hvcn1 ablation deregulates neutrophil pH p , leading to alkalinization in phagosomes with residual ROS production or to the early accumulation of V-ATPase on phagosomes that fail to mount an oxidative response. Hv1 channels therefore differentially regulate the pH p in neutrophils and macrophages, sustaining rapid acidification in macrophage phagosomes and maintaining a neutral pH in neutrophil phagosomes., (© 2014 Society for Leukocyte Biology.)

The vacuolar ATPase enzyme complex (V-ATPase) pumps protons across membranes, energized by hydrolysis of ATP. Extensive investigations on structural and biochemical features of these molecules have implied their importance in the physiological process. In this study, a full-length sequence encoding a vacuolar ATP synthase subunit ε-like protein of Clonorchis sinensis (CsATP-ε) was isolated from our cDNA library. The hypothetical 226 amino acid sequence shared 76% identity with ATP-ε proteins of Schistosoma japonicum and above 55% identity with ATP-ε proteins from human and other eukaryotes. Characteristic Asp₁₄₀ amino acid residues and seven B-cell epitopes were predicted in this sequence. The complete coding sequence of the gene was expressed in Escherichia coli. Recombinant CsATP-ε (rCsATP-ε) protein could be probed by anti-rCsATP-ε rat serum and C.sinensis-infected human serum in Western blotting experiment, indicating that it is an antigen of strong antigenicity. The high level of antibody titers (1:204,800) showed that CsATP-ε has a powerful immunogenicity. Both the increased level and the change trend of IgG1/IgG2a subtypes in serum showed that the rCsATP-ε can induce strong combined Th1/Th2 immune responses in rats and stimulate the immune response changes to the dominant Th2 from Th1 along with long time infection. The results of immunoblot and immunolocalization demonstrated that CsATP-ε was consecutively expressed at various developmental stages of the parasite, which was supported by real-time PCR analysis. In immunohistochemistry, CsATP-ε was localized on the intestine, vitellarium, and testicle of an adult worm and excretory bladder of metacercaria, implying that CsATP-ε may relate to energy intake and metabolism. This fundamental study would contribute to further researches that are related to growth and development and immunomodulation of C. sinensis.

Aims/hypothesis: The renin-angiotensin system (RAS) potentially has a role in the development of end-organ damage, and tissue RAS activation has been suggested as a risk factor for diabetic retinopathy. We have recently shown significant involvement of (pro)renin receptor ([P]RR) in retinal inflammation in a rodent model of early diabetes. In this study we aim to elucidate the (P)RR-associated pathogenesis of fibrovascular proliferation, a late-stage angiogenic complication in human diabetic retinopathy., Methods: Vitreous fluids from 23 eyes of patients with proliferative diabetic retinopathy (PDR) and 16 eyes of controls with non-diabetic, idiopathic macular diseases (macular hole and epiretinal membrane) were collected. Protein levels of soluble (P)RR were measured by ELISA, and immunofluorescence was performed to assess the localisation of (P)RR and related molecules in fibrovascular tissues from PDR eyes., Results: (P)RR immunoreactivity was detected in neovascular endothelial cells, colocalised with prorenin, phosphorylated extracellular signal-regulated kinase (ERK) and vascular endothelial growth factor (VEGF). Prorenin application to human retinal microvascular endothelial cells significantly upregulated mRNA expression of VEGF, especially the VEGF165 isoform, which was abolished by (P)RR or ERK signalling blockade. Proteases known to cleave (P)RR, including furin, were positive in endothelial cells in fibrovascular tissues. Protein levels of soluble (P)RR in vitreous fluids were higher in PDR eyes than in non-diabetic control eyes, and correlated significantly with vitreous prorenin and VEGF levels and the vascular density of fibrovascular tissues., Conclusions/interpretation: Our data using human samples provide the first evidence that (P)RR is associated with angiogenic activity in PDR.

Adiponectin (APN), a cytokine constitutively produced in fat tissue, has been shown to exert anti-inflammatory effects in various disease models. While the influence of APN on monocytic cells has been extensively studied in vitro, little is known about its role in T cells. In this study, we show that while <10% of human peripheral blood T cells express adiponectin receptors (AdipoRs) on their surface, most T cells store AdipoRs in intracellular compartments. AdipoRs colocalized with immune regulatory molecules CTLA-4 and TIRC7 within clathrin-coated vesicles. After stimulation, the expression of adiponectin receptor 1 (AdipoR1) and AdipoR2 was upregulated on the surface of antigen-specific T cells, as determined by tetramer or CD137 staining, and AdipoR1 and AdipoR2 coexpressed with CTLA-4. Addition of APN resulted in a significant diminution of antigen-specific T-cell expansion. Mechanistically, APN enhanced apoptosis and inhibited proliferation of antigen-specific T-cell lines. Further, APN directly inhibited cytokine production in response to antigen stimulation. In line with the in vitro data, APN-deficient (knockout, KO) mice had higher frequencies of CD137(+) T cells upon Coxsackie B virus infection. Altogether, our data suggest that APN is a novel negative T-cell regulator. In contrast to the CTLA-4 ligand B7 only expressed on APCs, APN is abundant in human plasma., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cross-presentation of IgG-containing immune complexes (ICs) is an important means by which dendritic cells (DCs) activate CD8(+) T cells, yet it proceeds by an incompletely understood mechanism. We show that monocyte-derived CD8(-)CD11b(+) DCs require the neonatal Fc receptor for IgG (FcRn) to conduct cross-presentation of IgG ICs. Consequently, in the absence of FcRn, Fcγ receptor (FcγR)-mediated antigen uptake fails to initiate cross-presentation. FcRn is shown to regulate the intracellular sorting of IgG ICs to the proper destination for such cross-presentation to occur. We demonstrate that FcRn traps antigen and protects it from degradation within an acidic loading compartment in association with the rapid recruitment of key components of the phagosome-to-cytosol cross-presentation machinery. This unique mechanism thus enables cross-presentation to evolve from an atypically acidic loading compartment. FcRn-driven cross-presentation is further shown to control cross-priming of CD8(+) T-cell responses in vivo such that during chronic inflammation, FcRn deficiency results in inadequate induction of CD8(+) T cells. These studies thus demonstrate that cross-presentation in CD8(-)CD11b(+) DCs requires a two-step mechanism that involves FcγR-mediated internalization and FcRn-directed intracellular sorting of IgG ICs. Given the centrality of FcRn in controlling cross-presentation, these studies lay the foundation for a unique means to therapeutically manipulate CD8(+) T-cell responses.

The vacuolar-type proton pump ATPase (V-ATPase) plays several pivotal roles in the acidification of diverse intracellular compartments and the extracellular environment. The a subunit isoforms a1, a2, and a3, constituting the membrane-embedded section, are expressed in various tissues, and they are involved in the regulation of subcellular localization and activity of the holocomplex. Therefore, the characterization of their properties is indispensable for dissection of the physiological roles of the V-ATPase in highly differentiated cells. In this study, we report the production and characterization of chicken monoclonal antibodies (MAbs) against these mouse a1, a2 and a3 subunit isoforms. These MAbs are shown to be suitable for both immunoblotting and immunofluorescence analysis. The MAbs obtained in this study are useful in understanding the pathological basis of V-ATPase dysfunction.

In mammalian reproduction, two immunologically disparate entities, the mother and her fetus, co-exist in close proximity and mutually tolerate each other. The maternal immune system plays a major contributing role in the reproductive outcome. A coordinated set of immunological events takes place between the maternal and fetal cells to ensure fetal survival. Among these, cytokines secreted by proximal maternal immune cells as well as fetal trophoblast cells play a major role in feto-maternal tolerance. In this review, we describe the role of the vacuolar ATPase (and more specifically the a2 isoform, a2V-ATPase) in controlling the expression of these vital cytokines. a2V-ATPase is a key enzyme that controls the acidification of intracellular vesicles and the extracellular environment, processes that play a major role in cellular function. The localization of a2V-ATPase in tissues and immune cells of the reproductive tract which are essential for pregnancy will be described. Information will be provided on the role of a2V-ATPase on aspects of cell development in pregnancy, from fertilization to implantation and fetal growth. Particular emphasis will be placed on the role of a2V-ATPase in (a) regulating parts of the cytokine network at the implantation site and (b) attenuating the potentially harmful maternal immune response against trophoblast cells., (Copyright (c) 2009 Elsevier Ireland Ltd. All rights reserved.)

Suaeda salsa L. is a halophytic species that is well adapted to high salinity. In order to understand its salt tolerance mechanism, we examined the growth and vacuolar H(+)-ATPase (V-ATPase) response to NaCl within the shoots and roots. The growth of shoots, but not roots, was dramatically stimulated by NaCl. Cl(-) and Na(+) were mainly accumulated in shoots. V-ATPase activity was significantly increased by NaCl in roots and especially in shoots. Interestingly, antisera ATP95 and ATP88b detected three V(1) subunits (66, 55 and 36 KDa) of V-ATPase only in shoots, while an 18 kDa V(0) subunit of V-ATPase was detected by both antisera in shoots and roots. It suggested that the tissue-specific characteristics of V-ATPase were related to the different patterns of growth and ion accumulation in shoots and roots of S. salsa.

H+ transport driven by V H+-ATPase was found in membrane fractions enriched with ER/PM and Golgi/Golgi-like membranes of Saccharomyces cerevisiae efficiently purified in sucrose density gradient from the vacuolar membranes according to the determination of the respective markers including vacuolar Ca2+-ATPase, Pmc1::HA. Purification of ER from PM by a removal of PM modified with concanavalin A reduced H+ transport activity of P H+-ATPase by more than 75% while that of V H+-ATPase remained unchanged. ER H+ ATPase exhibits higher resistance to bafilomycin (I50=38.4 nM) than Golgi and vacuole pumps (I50=0.18 nM). The ratio between a coupling efficiency of the pumps in ER, membranes heavier than ER, vacuoles and Golgi is 1.0, 2.1, 8.5 and 14 with the highest coupling in the Golgi. The comparative analysis of the initial velocities of H+ transport mediated by V H+-ATPases in the ER, Golgi and vacuole membrane vesicles, and immunoreactivity of the catalytic subunit A and regulatory subunit B further supported the conclusion that V H+-ATPase is the intrinsic enzyme of the yeast ER and Golgi and likely presented by distinct forms and/or selectively regulated.

Natural CD25(+)CD4(+) regulatory T cells (Treg) are essential for self-tolerance and for the control of T cell-mediated immune pathologies. However, the identification of Tregs in an ongoing immune response or in inflamed tissues remains elusive. Our experiments indicate that TIRC7, T cell immune response cDNA 7, a novel membrane molecule involved in the regulation of T lymphocyte activation, identifies two Treg subsets (CD25(low)TIRC7(+) and CD25(high)TIRC7(-)) that are characterized by the expression of Foxp3 and a suppressive activity in vitro and in vivo. We also showed that the CD25(low)TIRC7(+) subset represents IL-10-secreting Tregs in steady state, which is accumulated intratumorally in a tumor-bearing mice model. Blockade of the effect of IL-10 reversed the suppression imposed by the CD25(low)TIRC7(+) subset. Interestingly, these IL-10-secreting cells derived from the CD25(high)TIRC7(-) subset, both in vitro and in vivo, in response to tumoral Ags. Our present results strongly support the notion that, in the pool of natural Tregs, some cells can recognize foreign Ags and that this recognition is an essential step in their expansion and suppressive activity in vivo.

Immunocytochemistry performed on paraffin or cryosections is often hampered by poor morphology. Epoxy sections, in contrast, generally retain well-preserved tissue architecture. Immunocytochemistry, however, on epoxy-embedded sections is difficult due in part to the plastic itself and to the fixation conditions. Here, we present a technique for visualization of membrane proteins by immunocytochemistry on epoxy sections of kidneys fixed with glutaraldehyde without or with osmium post-fixation. Semithin sections were obtained from Epon 812-embedded mouse and rat kidney blocks. Before immunoperoxidase or immunofluorescence labeling, the sections were etched with the epoxy solvent, methanolic potassium hydroxide, followed by antigen retrieval using microwave heating. The sections were then treated with the primary antibody followed by secondary antibodies as usual. The distribution and expression patterns of a variety of membrane proteins, such as aquaporin (AQP)-1, AQP-2, and megalin, were identical to those observed by traditional immunocytochemical procedures on paraffin or cryosections. The advantages of our novel method include not only enhanced morphological quality but also the feasibility for investigators to visualize antigens of interest using archival specimens in Epon blocks.

The membrane protein T-cell immune response cDNA 7 (TIRC7) is transiently expressed in subsets of lymphocytes following antigen stimulation. The importance of TIRC7 in immune activation is demonstrated by the effect of antibodies directed against extracellular domains of TIRC7. In vitro targeting of TIRC7 inhibits proliferation and cytokine expression in human, mouse and rat lymphocytes, and these inhibitory effects have been associated with induction of cytotoxic T-lymphocyte antigen 4 mRNA and protein in the presence of TIRC7 antibodies. In vivo, anti-TIRC7 antibodies prevent kidney transplant rejection in rats and heart allograft rejection in mice. Treatment with an anti-TIRC7 antibody as monotherapy or in combination with TNFalpha blockade inhibits disease progression in collagen-induced arthritis. TIRC7 expression decreases in the peripheral blood of humans who have undergone cardiac transplant prior to clinical rejection, and is therefore a promising noninvasive tool for the prediction of rejection. Thus, targeting of TIRC7 may lead to the development of specific and effective therapeutic and diagnostic approaches by unifying relevant cellular and molecular responses in T- and B-cell subsets, and represents a promising new pathway for immune regulation in transplantation and autoimmune disease.

There is considerable evidence that osteoclasts are involved in the pathogenesis of juxta-articular bone destruction in rheumatoid arthritis. Vacuolar ATPases (V-ATPases), which are highly expressed in the ruffled border membrane of osteoclasts, play a central role in the process of bone resorption, and V-ATPase inhibitors are effective in preventing bone destruction in several animal models of lytic bone diseases. Here, we evaluated for the first time the effects of V-ATPase inhibition in rats with adjuvant-induced arthritis (AIA) using FR177995, a novel V-ATPase inhibitor. FR177995 completely inhibited H(+) transport driven by V-ATPase, but exerted no effect on the H(+) transport activities of F- and P-ATPase, indicating that FR177995 is a specific inhibitor of V-ATPase. FR177995 acted directly on osteoclastic bone resorption and equally inhibited in vitro bone resorption stimulated by IL-1, IL-6 or PTH. In addition, FR177995 dose-dependently reduced retinoic acid-induced hypercalcemia in thyroparathyroidectomized-ovariectomized rats. When FR177995 was administered to AIA rats once a day, the loss of femoral bone mineral density was significantly improved. Moreover, indicators of cartilage damage (arthritis score and glycosaminoglycan content in the femoral condyles) and inflammation parameters (paw swelling volume, erythrocyte sedimentation rate and plasma sialic acid level) were found to be unexpectedly ameliorated. These results strongly suggest that V-ATPase may be an interesting drug target in the treatment of rheumatoid arthritis.

Ab targeting of TIRC7 has been shown previously to inhibit T cell proliferation and Th1 lymphocyte-associated cytokine production. In this study, we demonstrate that Ab targeting of TIRC7 induces early cell surface expression of CTLA-4. The majority of stimulated CD4+ and CD8+ human T cells coexpress CTLA-4 and TIRC7. Similar to CTLA-4, TIRC7 rapidly accumulates at the site of Ag adhesion upon T cell activation. TIRC7 seems to colocalize with CTLA-4 in human T cells, and both molecules are associated with clathrin-coated vesicles, indicating they share intracellular transport systems. Moreover, Ab targeting of TIRC7 results in an early activation of CTLA-4 transcription. The inhibition of cell proliferation mediated by TIRC7 is dependent on CTLA-4 expression because the TIRC7-mediated inhibitory effects on cell proliferation and cytokine expression are abolished by Ab blockade of CTLA-4. Splenocytes obtained from CTLA-4-deficient mice are not responsive to TIRC7 Ab targeting. Thus, TIRC7 acts as an upstream regulatory molecule of CTLA-4 expression.

TIRC7 delivers essential signals during immune activation as antibodies targeting TIRC7 inhibit lymphocyte proliferation and Th1 cytokine expression in vitro and prolonged kidney and heart allograft survival in vivo. Immunohistochemical analysis of biopsy specimens from human renal allografts undergoing rejection despite treatment with Calcineurin inhibitors (CI) showed elevated TIRC7 expression. Accordingly, with a view to clinical application, we evaluated the therapeutic effect of a chimerized anti-TIRC7 mAb in combination with Tacrolimus (FK506) using a rat kidney transplantation model (DA to Lewis). The combination of sub-therapeutic doses of both compounds significantly (p<0.05) prolonged the median graft survival to 19.5 days compared to monotherapy with FK506 (median survival, 7d) or mAb against TIRC7 (7d). These results suggest a potential synergism of anti-TIRC7 mAb and FK506 action, which could be developed into a novel combination therapy in the clinic by lowering side effects of present CI treatment. Moreover, the identification of TIRC7 in graft infiltrating lymphocytes might serve as a diagnostic marker to detect allograft rejection.

TIRC7 is a cell surface molecule which is expressed in T and B lymphocytes and negatively regulates their function. Anti-TIRC7 specific monoclonal antibody (mAb) inhibited T cell memory response to recall antigens. Up-regulation of TIRC7 on lymphocytes from joint tissue of patients with Rheumatoid Arthritis (RA) and mice with collagen induced arthritis (CIA) suggested TIRC7 as a novel target to promote anti-inflammatory reaction. Anti-TIRC7 mAb administration significantly inhibited the induction and progression of CIA and the anti-collagen IgG1 and IgG2a antibody response. Combination therapy of anti-TIRC7 mAb and soluble TNF-alpha receptor demonstrated an increased inhibitory effect over the single compounds on CIA. The results demonstrate the therapeutic potential of TIRC7 targeting with mAb in diseases associated with exaggerated T and B cell responses.

In the epididymis and vas deferens, the vacuolar H(+)ATPase (V-ATPase), located in the apical pole of narrow and clear cells, is required to establish an acidic luminal pH. Low pH is important for the maturation of sperm and their storage in a quiescent state. The V-ATPase also participates in the acidification of intracellular organelles. The V-ATPase contains many subunits, and several of these subunits have multiple isoforms. So far, only subunits ATP6V1B1, ATP6V1B2, and ATP6V1E2, previously identified as B1, B2, and E subunits, have been described in the rat epididymis. Here, we report the localization of V-ATPase subunit isoforms ATP6V1A, ATP6V1C1, ATP6V1C2, ATP6V1G1, ATP6V1G3, ATP6V0A1, ATP6V0A2, ATP6V0A4, ATP6V0D1, and ATP6V0D2, previously labeled A, C1, C2, G1, G3, a1, a2, a4, d1, and d2, in epithelial cells of the rat epididymis and vas deferens. Narrow and clear cells showed a strong apical staining for all subunits, except the ATP6V0A2 isoform. Subunits ATP6V0A2 and ATP6V1A were detected in intracellular structures closely associated but not identical to the TGN of principal cells and narrow/clear cells, and subunit ATP6V0D1 was strongly expressed in the apical membrane of principal cells in the apparent absence of other V-ATPase subunits. In conclusion, more than one isoform of subunits ATP6V1C, ATP6V1G, ATP6V0A, and ATP6V0D of the V-ATPase are present in the epididymal and vas deferens epithelium. Our results confirm that narrow and clear cells are well fit for active proton secretion. In addition, the diverse functions of the V-ATPase may be established through the utilization of specific subunit isoforms. In principal cells, the ATP6V0D1 isoform may have a physiological function that is distinct from its role in proton transport via the V-ATPase complex.

The ubiquitous multisubunit vacuolar-type proton pump (H+- or V-ATPase) is essential for acidification of diverse intracellular compartments. It is also present in specialized forms at the plasma membrane of intercalated cells in the distal nephron, where it is required for urine acidification, and in osteoclasts, playing an important role in bone resorption by acid secretion across the ruffled border membrane. It was reported previously that, in human, several of the renal pump's constituent subunits are encoded by genes that are different from those that are ubiquitously expressed. These paralogous proteins may be important in differential functions, targeting or regulation of H+-ATPases. They include the d subunit, where d1 is ubiquitous whereas d2 has a limited tissue expression. This article reports on an investigation of d2. It was first confirmed that in mouse, as in human, kidney and bone are two of the main sites of d2 mRNA expression. d2 mRNA and protein appear later during nephrogenesis than does the ubiquitously expressed E1 subunit. Mouse nephron-segment reverse transcription-PCR revealed detectable mRNA in all segments except thin limb of Henle's loop and distal convoluted tubule. However, with the use of a novel d2-specific antibody, high-intensity d2 staining was observed only in intercalated cells of the collecting duct in fresh-frozen human kidney, where it co-localized with the a4 subunit in the characteristic plasma membrane-enhanced pattern. In human bone, d2 co-localized with the a3 subunit in osteoclasts. This different subunit association in different tissues emphasizes the possibility of the H+-ATPase as a future therapeutic target.

A number of leukocyte surface molecules play an essential role during immune activation. Targeting of these molecules utilizing antibodies serves as a specific therapeutic approach for the treatment of a variety of human diseases. Antibodies targeting a number of leukocyte surface molecules were shown to induce tolerance to transplants in several animal models. A novel membrane molecule, T-cell immune response cDNA 7 (TIRC7), has been shown to be an essential protein in the regulation of lymphocyte activation. TIRC7 does not share any homology with other known membrane proteins expressed during the course of lymphocyte activation and does not belong to any of the known costimulatory, cytokine, chemokine or receptor families. TIRC7, a highly conserved protein across species, is expressed in immune tissues such as spleen, lymph nodes, and T and B lymphocytes. Antibodies against extracellular domains of TIRC7 prolong allograft survival in rat and mouse transplantation models. The prevention of rejection is mediated at least partially via induction of cytotoxic T lymphocyte antigen 4 (CTLA4) in T cells. Functional cellular assays utilizing TIRC7-deficient mice splenocytes show that TIRC7 does have an impact not only on T-cell, but also on B-cell response. Subtherapeutic amounts of FK506 and anti-TIRC7 monoclonal antibody prolong graft survival, suggesting synergistic effects with calcineurin inhibitors. Targeting TIRC7 with monoclonal antibody might serve as a promising therapeutic strategy for preventing allograft rejection in humans and treatment of other immune-related diseases. Acutely rejected human kidney allografts show strong expression of TIRC7 despite treatment with calcineurin inhibitors. Therefore, monitoring TIRC7 expression may facilitate an early diagnostic tool of acute rejection. TIRC7 seems to belong to a group of targets with dual roles in disease pathogenesis, so-called theranostics, which can be utilized to treat and diagnose diseases., ((c) 2005 Prous Science. All rights reserved.)

Embryonic-maternal signaling is vital to implantation. We have identified a cellular protein that indirectly regulates secretion of IL-1beta, a proinflammatory cytokine that is involved in this signaling process. Regeneration and tolerance factor is a V-ATPase protein that regulates ATP levels in a variety of cells including macrophages, which, in turn, regulates the P2X7 ligand-gated ion channel. As extracellular levels of ATP rise, the P2X7 receptor undergoes a change in permeability, which leads to the onset of apoptotic events and the release of IL-1beta. IL-1beta leads to local inflammation and vascularization, which is integral to establishing a successful implantation site.

The membrane protein T cell immune response cDNA 7 (TIRC7) was recently identified and was shown to play an important role in T cell activation. To characterize the function of TIRC7 in more detail, we generated TIRC7-deficient mice by gene targeting. We observed disturbed T and B cell function both in vitro and in vivo in TIRC7(-/-) mice. Histologically, primary and secondary lymphoid organs showed a mixture of hypo-, hyper-, and dysplastic changes of multiple lymphohemopoietic compartments. T cells from TIRC7(-/-) mice exhibited significantly increased proliferation and expression of IL-2, IFN-gamma, and IL-4 in response to different stimuli. Resting T cells from TIRC7(-/-) mice exhibited decreased CD62L, but increased CD11a and CD44 expression, suggesting an in vivo expansion of memory/effector T cells. Remarkably, activated T cells from TIRC7(-/-) mice expressed lower levels of CTLA-4 in comparison with wild-type cells. B cells from TIRC7-deficient mice exhibited significantly higher in vitro proliferation following stimulation with anti-CD40 Ab or LPS plus IL-4. B cell hyperreactivity was reflected in vivo by elevated serum levels of various Ig classes and higher CD86 expression on B cells. Furthermore, TIRC7 deficiency resulted in an augmented delayed-type hypersensitivity response that was also reflected in increased mononuclear infiltration in the skin obtained from TIRC7-deficient mice food pads. In summary, the data strongly support an important role for TIRC7 in regulating both T and B cell responses.

Background: Vacuolar H+-ATPases are large protein complexes of more than 700 kDa that acidify endomembrane compartments and are part of the secretory system of eukaryotic cells. They are built from 14 different (VHA)-subunits. The paper addresses the question of sub-cellular localisation and subunit composition of plant V-ATPase in vivo and in vitro mainly by using colocalization and fluorescence resonance energy transfer techniques (FRET). Focus is placed on the examination and function of the 95 kDa membrane spanning subunit VHA-a. Showing similarities to the already described Vph1 and Stv1 vacuolar ATPase subunits from yeast, VHA-a revealed a bipartite structure with (i) a less conserved cytoplasmically orientated N-terminus and (ii) a membrane-spanning C-terminus with a higher extent of conservation including all amino acids shown to be essential for proton translocation in the yeast. On the basis of sequence data VHA-a appears to be an essential structural and functional element of V-ATPase, although previously a sole function in assembly has been proposed., Results: To elucidate the presence and function of VHA-a in the plant complex, three approaches were undertaken: (i) co-immunoprecipitation with antibodies directed to epitopes in the N- and C-terminal part of VHA-a, respectively, (ii) immunocytochemistry approach including co-localisation studies with known plant endomembrane markers, and (iii) in vivo-FRET between subunits fused to variants of green fluorescence protein (CFP, YFP) in transfected cells., Conclusions: All three sets of results show that V-ATPase contains VHA-a protein that interacts in a specific manner with other subunits. The genomes of plants encode three genes of the 95 kDa subunit (VHA-a) of the vacuolar type H+-ATPase. Immuno-localisation of VHA-a shows that the recognized subunit is exclusively located on the endoplasmic reticulum. This result is in agreement with the hypothesis that the different isoforms of VHA-a may localize on distinct endomembrane compartments, as it was shown for its yeast counterpart Vph1.

T cell immune response c-DNA (TIRC7) is up-regulated during the early stages of T-cell activation in response to alloantigens. In this study, we analyzed the effects of newly developed monoclonal antibodies (mAb) against TIRC7 in acute cardiac allograft rejection. Fully vascularized heterotopic allogeneic heart transplantation was performed in mice across a full-mismatch barrier (C57Bl/10 into CBA). Recipients received seven injections (day 0-7) of a novel anti-TIRC7 mAb or remained untreated. Graft survival, histology and ex vivo lymphocyte functions were tested. Targeting of TIRC7 with an anti-TIRC7 mAb diminishes lymphocyte infiltration into grafts resulting in delay of morphological graft damage and prolongation of allograft survival. The lymphocytes from anti-TIRC7 mAb-treated animals exhibit hypo-responsiveness without evidence of lymphocyte depletion against the donor allo-antigens. Proliferation and expression of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) were down-regulated while interleukin-4 (IL-4) and IL-10 expression were spared. Moreover, anti-TIRC7 mAb enhanced up-regulation of CTLA-4 expression but suppressed up-regulation of CD25 on stimulated lymphocytes in vitro and in vivo. Ligation of TIRC7 has important effects on the regulation of co-stimulatory signaling pathways associated with suppressing of T-cell activation. Targeting of TIRC7 may therefore provide a novel therapeutic approach for modulating T cell immune responses during organ transplantation.

The vacuolar ATPase is a multifunctional enzyme that consists of several subunits. Subunit B is part of the catalytic domain of the enzyme and is present in two isoforms in fish as well as in mammals. Possibly, these two isoforms - vatB1 (kidney isoform) and vatB2 (brain isoform) - serve different functions. A localization of the two isoforms was attempted in swimbladder gas gland cells of the European eel Anguilla anguilla by immunohistochemistry. Two antibodies were produced by immunization of rabbits with synthetic peptides. Specificity of the antibodies, on the one hand, an isoform-specific antibody for vatB1 and, on the other hand, an antibody that recognizes both isoforms (vatB1 and vatB2), was confirmed by western blot analysis using recombinant proteins produced in a bacterial expression system. The immunohistochemical localization with the antibody directed against both isoforms of the B subunit revealed a positive staining in apical membranes of swimbladder gas gland cells as well as in the basolateral membranes. Significant staining was observed in vesicles located near the apical membrane. Staining with the vatB1-specific antibody resulted in a similar picture in the apical region of the cells. In contrast to the staining with the first antibody, only a poor signal was observed in the basal region. The nature of the vesicles in the apical region of the gas gland cells was determined by using an antibody directed against surfactant protein D.

An important goal of cancer immunology is the identification of antigens associated with tumor destruction. Vaccination with irradiated tumor cells engineered to secrete granulocyte/macrophage colony-stimulating factor (GM-CSF) generates potent, specific, and long-lasting antitumor immunity in multiple murine tumor models. A phase I clinical trial of this vaccination strategy in patients with advanced melanoma demonstrated the consistent induction of dense CD4(+) and CD8(+) T lymphocyte and plasma cell infiltrates in distant metastases, resulting in extensive tumor destruction, fibrosis, and edema. Antimelanoma antibody and cytotoxic T cell responses were associated with tumor cell death. To characterize the targets of these responses, we screened an autologous cDNA expression library prepared from a densely infiltrated metastasis with postvaccination sera from a long-term responding patient. High-titer IgG antibodies detected ATP6S1, a putative accessory unit of the vacuolar H(+)-ATPase complex. A longitudinal analysis of this patient revealed an association between the vaccine-induced increase in antibodies to ATP6S1 and tumor destruction. Three additional vaccinated melanoma patients and three metastatic non-small cell lung carcinoma patients vaccinated with autologous GM-CSF-secreting tumor cells similarly showed a correlation between humoral responses to ATP6S1 and tumor destruction. Moreover, a chronic myelogenous leukemia patient who experienced a complete remission after CD4(+) donor lymphocyte infusions also developed high-titer antibodies to ATP6S1. Lastly, vaccination with GM-CSF-secreting B16 melanoma cells stimulated high-titer antibodies to ATPS1 in a murine model. Taken together, these findings demonstrate that potent humoral responses to ATP6S1 are associated with immune-mediated destruction of diverse tumors.