Low Molecular Weight Antigens Research Articles

A GROUP OF LOW MOLECULAR WEIGHT (

Heat-killed Mycobacterium tuberculosis (Mtb) H37Rv–derived antigens were purified by electroelution, and their efficacy was evaluated in conferring protection against Tb in guinea pigs in order to identify those protein antigens as a subunit vaccine. A total sonicate extract of Mtb H37Rv (MTSE) was prepared by growing bacteria on Lowenstein–Jensen (LJ) solid medium to its mid-log phase, harvested, and subjected to ultrasonication. Fractions of MTSE were resolved on 10% polyacrylamide gel electrophoresis into as high (HMW, >60 kDa), middle (MMW, 31-59 kDa), and low molecular weight (LMW, <30 kDa) antigens and were purified by electroelution. The protection efficacy of each purified fraction was assessed in a placebo-controlled study by injecting them separately into a group of two animals. After two booster doses, animals were challenged on day 84, and autopsy of all animals was performed on day 126. On autopsy, animals were scored for formation of tubercles in lungs and their dissemination to organs such as spleen, liver, kidney, and lymph nodes. Viable counts (determined as colony forming unit or CFU) of lung and spleen from each animal were performed on LJ solid medium. Results of this study show that CFU of animals that received LMW antigens were significantly low with respect to control animals when compared to MMW and HMW. In conclusion, LMW of Mtb has greater potential in prevention of tubercle formation and thus could be viewed as a possible vaccine candidate in designing alternative subunit vaccines.

Colonization by Helicobacter pylori of leprosy patients in Spain: immunomodulation to low molecular weight antigens of H. pylori

We studied the prevalence of Helicobacter pylori in patients with leprosy and the effects of co-infection on the immune response to Helicobacter antigens in the polar groups of leprosy (lepromatous and tuberculoid). We showed that there is no difference in the prevalence of H. pylori in patients with leprosy as compared to a non-leprosy population. We also demonstrated that the immune response to low molecular weight H. pylori antigens (35, 26 and 19 kDa) differs in patients with lepromatous as compared to those with tuberculoid leprosy. In lepromatous leprosy, we show that there is a higher prevalence of the 35 and 26 kDa antigens, but a lower prevalence of the 19 kDa antigen. These immunological results are consistent with previous histopathological studies illustrating a more severe gastrointestinal inflammation in lepromatous patients; importantly, a response to the 35 kDa antigen is recognized as a marker for the development of ulcerative disease.

Simulation of perforated rectangular cantilever immunosensor for estimation of bacterial pathogens

Micro fabricated and multilayered perforated cantilever beam immunosensor was modeled using CoventorWare for the estimation of bacterial antigens of Bacillus Anthrax, Pseudomonas aeruginosa, Coryne Bacterium Diptheria and Treponema pallidum. A rectangular cantilever beam with perforations was simulated with dimensions as length-200 µm, width-10 µm and thickness-0.5 µm. Each perforation is rectangular with length-10 µm, width-5 µm and thickness-0.5 µm. The theoretical and FEM simulations were carried out with five immunoglobulin antibodies, IgA, IgD, IgE, IgG and IgM for the estimation of bacterial antigens. The effect of perforation in cantilever beam and molecular size of antibody and antigen on the performance of the sensor has been studied. The cantilever beam without perforation showed a deflection of 1.8 e + 02 µm whereas the cantilever beam with perforation showed an increase deflection of 1.9 e + 02 µm. With IgG, the difference between analytical and simulation values is positive and low especially with low molecular weight antigens Pseudomonas aeruginosa and Treponema pallidum. The low molecular weight IgG influences the antigen-antibody interaction more fvourably. The simulated perforated rectangular cantilever beam with IgG antibody is a more promising model for the fabrication of a sensor for the estimation of highly motile Pseudomonas aeruginosa and Treponema pallidum.

Open sandwich-based immuno-transistor for label-free and highly sensitive detection of low molecular weight antigens

Open sandwich-based immuno-transistor for label-free and highly sensitive detection of low molecular weight antigens

Adenovirus Virion Stability and the Viral Genome: Size Matters

Ongoing investigations into the fundamental aspects of virus biology continue to provide crucial insights for the development of more effective virus-based therapeutics. Within the field of gene therapy, these discoveries have translated into virus vectors with improved tissue tropism, prolonged transgene expression, and enhanced safety. However, radical modifications to the viral genome or capsid constituents can have marked effects on virion stability. In particular, recent insights into the role of viral genomes in contributing to the physical stability of the virion highlight the need to reconsider this heretofore underappreciated aspect of vector design. Here we discuss the impact of the genome on virion stability, with particular emphasis on the development of adenovirus (Ad)-based vectors.

Open Sandwich-Based Immuno-Transistor for Label-Free and Noncompetitive Detection of Low Molecular Weight Antigen

In this study, we proposed a new detection method, open sandwich-based immuno-field effect transistor (OS-FET) for label-free and noncompetitive detection of low molecular weight antigen. The principle of OS-FET is based on the detection of intrinsic molecular charges caused by the small antigen-dependent interchain interaction of separated V(L) and V(H) chains from a single antibody variable region using the field effect. Introducing V(H) chain and small antigen bisphenol A into the OS-FET with the immobilized V(L) chain on the gate, we could detect electrically and directly the binding of bisphenol A by V(H) and V(L) chains. Although the detection limit of OS-FET was 1 nM to detect bisphenol A for the standard deviation of control signal, the addition of isothiocyanobenzyl-EDTA with negative charges to the V(L) chain enhanced the detection limit to 1 pM. We could directly transduce the charge density changes based on the capture of target on the gate into the electrical signals using the OS-FET. The platform based on the FETs is suitable for a label-free, noncompetitive, and quantitative detection system for small antigen analysis in environmental, food, and clinical research.

Effect of gold nanoparticles on the respiratory activity of peritoneal macrophages

This work was undertaken to investigate the effect of antigen-conjugated gold nanoparticles on the functional activity of cells of the reticuloendothelial system. Experiments using rat and mouse peritoneal cells assessed the dynamics of the total respiratory activity of macrophages and the activity of mitochondrial dehydrogenases on exposure to colloidal gold conjugates of high- and lowmolecular-weight antigens. Both gold nanoparticles and their antigen conjugates stimulated the respiratory activity of the macrophages and the activity of macrophage mitochondrial enzymes. This stimulation may be an essential factor determining the adjuvant properties of colloidal gold, found by us in prior work.

Antibodies in Cold Stressed Mice Recognize a Surface Protein in Toxoplasma gondii Tachyzoites

Physical or psychological stressors are known to have significant consequences for immune function and the outcome of disease in human and animal models. In mice, cold water stress (CWS) has been shown to delay control of acute infection and reactivation of latent infections. Increased levels of parasite-specific IgG and IgM antibodies are observed when CWS is applied in the chronic phase. The present study examined the effects of a physical stressor, CWS, on tachyzoites antigens of Toxoplasma gondii, with particular emphasis on a low molecular weight antigen, 5 kDa, which seems to be recognized by antibodies from mice subjected to CWS in the chronic phase. This antigen is not recognized by antibodies from infected mice not subjected to CWS. Sera obtained from stressed and infected (CWS + INF) mice subjected to CWS during the chronic phase (CWS + INF + CWS) were used to harvest anti-5-kDa antibodies for immunolocalization studies. Tachyzoite lysate preparations were electrophoretically separated and transferred to nitrocellulose membranes. Strips of nitrocellulose containing tachyzoite antigens in the 4-10-kDa range were used to select for anti-5-kDa antibodies. Harvested anti-5-kDa localized this antigen on the surface of tachyzoites. This antigen was not present in bradyzoite preparations. Treatment with phosphatidylinositol-specific phospholipase C showed this antigen was not anchored to the cell membrane through glycosyl-phosphatidylinositol. Strong antibody responses in stressed animals during the chronic phase are associated with parasite reactivation. The 5-kDa antigen constitutes a unique immunogenic component of T. gondii, with significant diagnostic potential for identifying reactivation of latent infections.

Brugia malayi Adult Low Molecular Weight IgG4-Reactive Antigens Induce Differential Cytokine Response in Lymphocytes of Endemic Normal and Asymptomatic Microfilariae Carriers In Vitro

To characterize putatively protective immune response in bancroftian filariasis, Th1/Th2 cytokine profile induced in peripheral blood mononuclear cells (PBMCs) of endemic normal (EN) and asymptomatic microfilaremic (ASM) individuals were studied using different molecular weight fractions of Brugia malayi adult soluble antigens (BmA), which are differentially recognized by IgG4 antibodies present in their sera. Infection free and putatively immune individuals living in a filaria endemic area were identified and included in the present study as EN only after careful longitudinal follow up for three years. It was observed that the low molecular weight antigens present in Fr4 and Fr5 induced differential cytokine response; EN individuals showed a strong Th1 bias whereas ASM individuals showed a strong Th2 bias even though both the groups produced Th1 cytokines, albeit of different quantity, when a nonhelminthic antigen like H37Rv whole cell lysate was used. Since antigens present in Fr5 induced a highly polarized response, they should be examined for their diagnostic potential in lymphatic filariasis.

Three surface antigens dominate the mucosal antibody response to gastrointestinal L3-stage strongylid nematodes in field immune sheep

Although gastrointestinal nematode parasites are a major human and veterinary health problem, little is known about how the host is sometimes able to mount an effective immune rejection response. In previous work, we identified a carbohydrate larval surface antigen (CarLA) as the target of mucosal antibodies that can elicit rejection of Trichostrongylus colubriformis L3s in sheep. Here we characterise the natural mucosal antibody responses to L3s from three major strongylid gastrointestinal parasites of sheep, Trichostrongylus colubriformis, Haemonchus contortus and Teladorsagia circumcincta. The mucosal antibody repertoire of naturally field-immune sheep was displayed on bacteriophage as single-chain antibodies (scFvs) and phage were selected for the ability to bind to the surface of living L3s of the three nematode species. All nematode-binding scFvs were found to recognize one of three different antigen classes that are each found in the three strongylid species. These three antigen classes appear to represent all of the major antigens recognized on Western blots by pooled mucosal antibodies from field-immune sheep. One of the antigen classes is a heterogeneous, high molecular weight molecule that is protease-sensitive. The scFvs recognizing this surface antigen also recognize a similar antigen in all strongylids tested. A second antigen class is a protease-insensitive, low molecular weight antigen found only in sheaths and scFvs recognizing this antigen cross-react with a similar molecule found in all strongylids tested. The third surface antigen class is CarLA and all of the anti-CarLA scFvs obtained from the field-immune sheep repertoire were specific to L3s of only one species and often recognized only a subset of the worms. Thus three different L3-stage surface antigens, two that lack a protein component, dominate the natural mucosal antibody response to L3-stage gastrointestinal strongylid nematodes in sheep.

Leishmania amazonensis: Humoral response to amastigote excreted-antigens in murine leishmaniasis

With the purpose of studying the antigenic role that factors excreted by Leishmania amastigotes might have during murine infection, immunoblots were carried out with sera from C57BL/6 and BALB/c mice infected with two strains of Leishmania (L.) amazonensis, NR and IFLA/BR. Both strains differ widely in virulence in BALB/c mice. BALB/c but not C57BL/6 sera recognized several excretion products. The excreted antigens showed a strong response towards IgG1 and IgG2a isotypes whilst they reacted only weakly against IgG2b and IgG3. A low-molecular weight antigen (about 20kDa) excreted by both Leishmania strains was strongly recognized by IgG1 from BALB/c mice sera infected with IFLA/BR, the most virulent strain. Sera from NR infected mice were incapable of recognizing this antigen in spite of its presence in NR excreted products. The results indicate that the humoral immune response to excreted antigens of amastigotes depends on both the host genetic background and the parasite strain.

Western Blot Analysis of the IgG-Antibody Response to Acid- Glycine-Extracted Antigens from Campylobacter fetus subsp. fetus and C. jejuni in Naturally Infected Sheep

IgG-antibody response in aborting sheep and in apparently healthy sheep in a flock against acidglycine- extracted antigens from three strains for each C. fetus subsp. fetus and C. jejuni were analysed by Western blot. One strain of C. fetus subsp. fetus was isolated from aborting sheep. Western blot analysis of the sera revealed the presence of IgG antibody binding to the common antigens including proteins with the Mw of 63 kDa and 54 kDa in extracts from both C. fetus subsp. fetus and C. jejuni strains. In addition, IgG antibodies in sera from aborting sheep reacted more strongly with the antigens from C. fetus subsp. fetus strains with Mw of approximately 100, 95 and 86.5 kDa than those of apparently healthy sheep. The binding profile of the antibodies with these antigens appeared to be unique for each C. fetus subsp. fetus strain. On the other hand, IgG antibodies only in sera from aborting sheep recognized strongly the antigens of each C. fetus subsp. fetus strain at the Mw ranged from approximately 26 to 22 kDa. However, the antigenic components between 26 and 22 kDa were not detectable in coomassie blue stained gel and thought to have non-protein nature. These low molecular weight antigens of C. fetus subsp. fetus may be related to a recent infection in aborting sheep. These observations indicate that such speciesspecific antigens or conjugated protein antigens could be used for improving the specificity of the serological tests to detect C. fetus antibodies in sheep sera, and may be the candidates for subunit vaccines against ovine abortion.

Humoral response to low molecular weight antigens of Mycobacterium tuberculosis by tuberculosis patients and contacts

Much effort has been devoted to the identification of immunologically important antigens of Mycobacterium tuberculosis and to the combination of target antigens to which antibodies from serum of tuberculous patients could react specifically. We searched for IgG antibodies specific for antigens of 45 to 6 kDa obtained after sonication of the well-characterized wild M. tuberculosis strain in order to detect differences in the antibody response to low molecular weight antigens from M. tuberculosis between patients with pulmonary tuberculosis and contacts. Specific IgG antibodies for these antigens were detected by Western blot analysis of 153 serum samples collected from 51 patients with confirmed pulmonary tuberculosis. Three samples were collected from each patient: before therapy, and after 2 and 6 months of treatment. We also analyzed 25 samples obtained from contacts, as well as 30 samples from healthy individuals with known tuberculin status, 50 samples from patients with other lung diseases and 200 samples from healthy blood donors. The positive predictive value for associated IgG reactivity against the 6-kDa and 16-kDa antigens, 6 and 38 kDa, and 16 and 38 kDa was 100% since simultaneous reactivity for these antigens was absent in healthy individuals and individuals with other lung diseases. This association was observed in 67% of the patients, but in only 8% of the contacts. The humoral response against antigens of 16 and 6 kDa seems to be important for the detection of latent tuberculosis since the associated reactivity to these antigens is mainly present in individuals with active disease.

Isolates of Trichuris muris elicit different adaptive immune responses in their murine host

The J and S isolates of Trichuris muris have different infection profiles in C57BL/6 mice; J worms are expelled, S worms survive to chronicity. Building on this, the ability of the J and S isolates to survive, and the quality of the immune response induced was explored in three different strains of mouse. The resistant BALB/c mouse mounted a strong Th2 response against both isolates, which were quickly expelled. The susceptible AKR host mounted a Th1 response and retained both isolates. Despite equivalent worm exposure, mesenteric lymph node cells from AKR mice infected with the S isolate produced significantly higher levels of IL-12 and the intestinal mastocytosis was reduced. IgG1 and IgG2a from S-infected AKR mice recognized low molecular weight antigens not recognized by J-infected mice. Differential expulsion kinetics was observed in the slower-responding C57BL/6 strain; J worms were expelled but S isolate worms were retained. Survival of the S isolate was again associated with elevated IL-12 and decreased Th2 responses. In resistant mouse strains, the outcome of infection is thus dominantly influenced by host genetics. However, in the slower-responding host, isolate-derived factors may play a role in shaping the quality of the adaptive immune response, thus influencing parasite survival.

Highly sensitive and interference-free simultaneous detection of two polycyclic aromatic hydrocarbons at parts-per-trillion levels using a surface plasmon resonance immunosensor

A novel surface plasmon resonance (SPR)-based flow immunosensor is fabricated for simultaneous detection of benzo(a)pyrene (BaP), a potential endocrine disrupter, and 2-hydroxybiphenyl (HBP), a carcinogen. Immunosensor chip for SPR imaging is fabricated using easily moldable, flexible, thin polydimethylsiloxane (PDMS) films. PDMS films having multi-microchannel structures are prepared by molding process and act as flow-cell module. With the use of multi-microchannel flow-cell systems, samples of as small as 300μl are enough for SPR analysis of two different compounds present in a mixed analyte solution simultaneously. The SPR immunosensor working with indirect competitive immunoreaction method is demonstrated to detect the low molecular weight antigens, BaP and HBP, at ppt (parts-per-trillion) levels. Monoclonal antibodies against BaP and HBP undergo competitive immunoreaction between the antigens, BaP and HBP, present in analyte solution and the antigen–protein conjugates immobilized on the SPR sensor chip. The SPR responses of the immunoreaction are followed to determine the concentration of BaP and HBP in mixed analyte solutions. The SPR immunosensor is demonstrated to detect BaP and HBP at the same time in the concentration range of 10ppt–300ppb and 10ppt–1000ppb, respectively, with a response time of approximately 15min. The SPR immunosensor shows negligible cross-sensitivity between the two analytes. Regeneration of the sensor chip for repeated use is attained with the use of pepsin as the eluent.

R-MC46 monoclonal antibody stimulates adhesion and phagocytosis by rat macrophages

In our previous experiments it was shown that R-MC46 monoclonal antibody (mAb), produced at our Institute, stimulated homotypic aggregation of rat granulocytes and production ofproinflammatory cytokines. The aim of this study was to examine antigen expression and function, recognized by R-MC46 mAb on macrophages. The expression of R-MC46 antigen on thymic and peritoneal macrophages was investigated using immunocytochemistry and flow cytometry methods. Its biochemical characterization was performed by Western blot. The ability of R-MC46 mAb to modulate adhesion and phagocytosis by macrophages was studied by using co-culture experiments with autologous thymocytes. R-MC46 mAb stained thymic macrophages more strongly than peritoneal macrophages. After in vivo treatment of peritoneal macrophages with Pristane, a significant up-regulation of the R-MC46 antigen expression was observed Western blot analysis showed that the mAb recognized a low molecular weight antigen of about 5.5 kDa. R-MC46 mAb significantly enhanced binding and phagocytosis of thymocytes by both thymic and peritoneal macrophages. These processes were completely blocked by WT.3 (anti-CD18) mAb. The stimulation of binding thymocyte to macrophages was higher with the use of thymic macrophages,while the phagocytosis of these cells was higher in the presence of peritoneal macrophages. R-MC46 mAb recognized a new molecule expressed by rat macrophages. The antigen is most probably involved in 82 integrin-mediated adhesion and phagocytosis, as well as proinflammatory functions of macrophages.

Immunoregulation in onchocerciasis: predominance of Th1-type responsiveness to low molecular weight antigens of Onchocerca volvulus in exposed individuals without microfilaridermia and clinical disease.

Chronic and generalized onchocerciasis is associated with suppression of the parasite-specific cellular responsiveness, while exposed individuals without parasitological and clinical evidence of infection (endemic normals) display prominent cellular reactivity to Onchocerca volvulus antigens (OvAg). In order to identify those parasite antigens which may account for this differential cellular responsiveness, total adult worm-derived OvAg were fractionated by means of preparative SDS-PAGE and blot-elution into 22 antigen fractions of continuously decreasing molecular weight. Peripheral blood mononuclear cells (PBMC) from microfilariae (mf)-positive onchocerciasis patients (n = 18) proliferated weakly in response to all OvAg fractions. In contrast, in vitro reactivity of PBMC from endemic normals (n = 9) was depressed in response to OvAg of mol. wt 200-30 kD only, while antigens of mol. wt < 30 kD induced vigorous proliferation in these individuals compared with the microfilaridermic patients (P < 0.05). Highest proliferative reactivity of cells from endemic normals was observed in response to OvAg of mol. wt 15-11 kD. Furthermore, these low mol. wt antigen fractions induced substantial production of IL-2 and interferon-gamma (IFN-gamma) in PBMC from endemic normals, but not in those from onchocerciasis patients. Cells from individuals of both groups secreted similar amounts of IL-5 in response to all OvAg fractions, with highest production again being induced by low mol. wt OvAg. In contrast, PBMC from onchocerciasis patients clearly produced more IL-10 than did cells from endemic normals. This augmented IL-10 production by PBMC from mf-positive individuals was not only observed after stimulation with OvAg fractions, but was measured in unstimulated control cultures as well. IFN-gamma-specific mRNA in antigen-stimulated PBMC from endemic normals appeared to be more prominent than in cells from onchocerciasis patients. However, mRNA transcripts of IL-10 and IL-13 were clearly present in patients, but were absent or inconsistently observed in endemic normals. Our results suggest that vigorous Th1-type cellular responsiveness encountered in endemic normals is restricted to low mol. wt antigens of O. volvulus, while such reactivity will not be present in mf-positive individuals. Furthermore, spontaneous production of high levels of IL-10 in onchocerciasis patients is likely to suppress Th1-type immunity, and thus may favour manifestation of chronic onchocerciasis. These traits of cellular immunity may contribute to the differential outcome of O. volvulus infection, the manifestation of clinical disease, and may also regulate the build up of acquired immunity in humans.

Sensitive and specific serodiagnosis of river blindness using Onchocerca ochengi antigens

Despite the large-scale application of ivermectin for the treatment of human onchocerciasis, this filarial disease has remained a public health hazard, requiring rapid and sensitive diagnostic methods for more efficient management. Several serological methods based on homologous native or recombinant diagnostic antigens have been developed. However, these antigens have remained scarce in the endemic areas. We report herein the preparation and evaluation of an Onchocerca ochengi total worm extract together with its low molecular weight (<31 kDa) and 14.4 kDa antigens for the diagnosis of human onchocerciasis. The 14.4 kDa antigen and the low molecular weight antigen fraction were identified by Western blot experiments. The antigens were employed for the detection of IgG4 in sera of 43 onchocerciasis patients, 35 normal Africans, 15 schistosomiasis patients, 11 ascariasis patients, 9 loaisis patients and 3 healthy Europeans. The diagnostic specificity values were 94.5, 87.5 and 100%, while the sensitivities were 90.7, 80 and 76.6% using the worm extract, low molecular weight antigen fraction and the 14.4 kDa antigen, respectively. We conclude that total worm extracts of O. ochengi can conveniently replace recombinant antigen cocktails in poor settings that do not have access to the latter.

Local immune responses in sensitized sheep following challenge infection with Teladorsagia circumcincta.

Sheep were sensitized by weekly infections with Teladorsagia circumcincta over a 9-week period. After a 12-week rest, sheep were divided into four groups and killed without challenge or 3, 5 and 10 days post challenge (DPC) with 50000 L3. Recovery of challenge larvae from abomasal scrapings was highest at 3 DPC while no parasites were recovered by 10 DPC. Abomasal lymph nodes (ALN) of challenged sheep were significantly larger at 5 DPC, coinciding with an increase in the proportion of CD4 T cells and a decrease in CD21+ cells, probably reflecting the loss of CD21 from terminally differentiated antibody secreting cells. A significant increase was observed in gammadelta-TCR+ cells at 3 DPC in the ALN, while their number slightly decreased in the abomasal tissues throughout the challenge period. The number of tissue eosinophils was dramatically increased after challenge compared with the unchallenged controls, with a peak at 3 DPC, coinciding with the peak in larval recovery. CD4+ cells significantly increased in the abomasal tissues at 5 DPC, while no changes in globule leucocytes were observed until 10 DPC. Antibody-secreting cell probes (ASC-probes) generated from the ALN showed highest reactivity against larval antigens at 5 DPC. This reactivity was predominantly directed against regions between 90 and 100 kDa and 30-35 kDa in the L3 preparation and lower molecular weight antigens in the L4. No reactivity was observed against the adult extract. The 30-35 kDa antigen seemed to exist as a high molecular weight complex in L3 homogenate and was not susceptible to protease K treatment, suggesting it may be non-protein in nature.

Differentiation of human γδ T cells towards distinct memory phenotypes

Vγ9/Vδ2 T cells comprise a small population of peripheral T cells responding towards the low molecular weight antigen, ( E)-4-hydroxy-3-methyl-but-2-enyl-pyrophosphate (HMB-PP). HMB-PP-stimulated Vγ9/Vδ2 T cells proliferated, expressed CCL5/RANTES, and upregulated markers like CD16, CD25, CD69, and CD94, in the presence of either IL-15 or IL-21. Vγ9/Vδ2 T cells grown in the presence of IL-15 differentiated into an effector/memory population characterized by production of TNF-α, expression of CD45RO and CCR5, and lack of CD62L, CD81, and CCR7. In contrast, Vγ9/Vδ2 T cells grown with IL-21 differentiated into putative central memory CD45RO + T cells that did not produce TNF-α, IFN-γ, or IL-4, and maintained expression of CD62L, CD81, and CCR7.