Alloimmune responses may also be elicited during unprotected heterosexual intercourse, which is associated + and CD8+ T cell proliferative responses were elicited by stimulating PBMC with the partners' irradiated monocytes and compared with 3rd party unrelated monocytes, using the CFSE method. Significant increase in allogeneic proliferative responses was found in the CD4+ and CD8+ T cells to the partners' irradiated monocytes, as compared with 3rd party unrelated monocytes (p≤0.001). However, a significant decrease in proliferative responses, especially of CD8+ T cells to the partners' compared with 3rd party monocytes was consistent with tolerization, in both the heterosexual and homosexual partners (p<0.01). Examination of CD4+CD25+FoxP3+ regulatory T cells by flow cytometry revealed a significantly greater proportion of these cells in the homosexual than heterosexual partners practising unprotected sex (p<0.05). Ex vivo studies of infectivity of PBMC with HIV-1 showed significantly greater inhibition of infectivity of PBMC from heterosexual subjects practising unprotected compared with those practising protected sex (p = 0.02).The investigation was carried out in well-defined heterosexual and homosexual monogamous partners, practising unprotected sex and a heterosexual cohort practising protected sex. Allogeneic CD4+ and CD8+ T cell proliferative responses to the partners' unmatched cells and a minority may be tolerized. However, a greater proportion of homosexual rather than heterosexual partners developed CD4+CD25FoxP3+ regulatory T cells. These results, in addition to finding greater inhibition of HIV-1 infectivity in PBMC ex vivo in heterosexual partners practising unprotected, compared with those practising protected sex, suggest that allogeneic immunity may play a significant role in the immuno-pathogenesis of HIV-1 infection.Both heterosexual and homosexual monogamous partners practising unprotected sex develop allogeneic CD4 However, natural allogeneic tolerance can be equally robust, as is seen in maternal tolerance of the fetal paternal semi-allogeneic HLA. These two reciprocal mechanisms have occupied the central stage of immunology. The critical importance of mature DC in immunity and immature DC in tolerance has been well documented + T cells either immune responses, or tolerance identified by expression of CD25+FoxP3+ regulatory cells. Alloimmune responses may inhibit HIV-1 transmission, as has been documented in vertical transmission from mother to baby The present study was based on the hypothesis that allogeneic stimulation of human monocyte derived DC may elicit in CD4+ and CD8+ T cells stimulated by the partners' irradiated monocytes (one way MLR), compared with 3rd party unrelated monocytes. A small proportion of partners' cells however, showed tolerization of CD4+ and CD8+ T cells. Examination of CD4+CD25+FoxP3+ T regulatory cells in the two cohorts revealed a significantly greater proportion of these cells in the homosexual than heterosexual partners and they were associated with the CD4+ T cell proliferative responses. These immune responses must have been elicited by the partners' HLA stimulating lymphoid cells in the rectal, vaginal and/or penile mucosa. Comparison of heterosexual partners practising unprotected with those practising protected sex showed a smaller proportion of CD4+ T cells derived from the protected cohort being allo-immunized. Ex vivo HIV-1 infectivity studies of PBMC suggest that allogeneic immunized heterosexual subjects practising unprotected sex may have acquired some protection when compared with those practising protected sex.The first objective of this investigation was to study allogeneic responses in homosexual and heterosexual couples practising unprotected sex and to compare these between the two cohorts. The second objective was to identify immunological criteria that differentiated those practising unprotected and protected sex and may have affected infectivity by HIV-1. Both cohorts showed significant allogeneic proliferative responses of CD4+ T cells were >95% pure and the DC expressed cell-surface CD83, CD80, CD86 and CD40 molecules (data not presented). Allogeneic stimulation of CD4+ or CD8+ T cells elicited conventional proliferative responses  were examined and this was shown to be at 1∶10 DC∶CD4+ T cell ratio (data not presented). A time course of 1–7 days revealed that co-culture of DC with T cells for 7 days resulted in a progressive increase in T regs from 1.5(±0.1)% on day 0, to 3.4(±0.4)% on day 2 (p = 0.05), 5.1(±0.8)% on day 4 (p = 0.04), reaching a maximal increase of 8.3(±0.2)% on day 7 (p = 0.001) (+CD25+FoxP3+ T reg cells and 28.9(±1.1)% CD4+CD25+FoxP3− effector cells (not presented). Allogeneic (9.6±2.0%) compared with autologous stimulation (6.4±1.5%) of CD4+ T cells showed an increase in CD40L expression in T reg cells (p = 0.066), but a significant decrease in CD40L+ effector T cells, from 13.6±2.7 to 3.0±1.1% (p = 0.022)  ; represe= 0.022) ; represein vitro elicited both T cell proliferative and regulatory CD4+ T cells. In order to see if these findings could be translated to a human model, we studied homosexual monogamous partners practising unprotected sex for at least 1 year  partners showing higher MLR than those with 3rd party cells were classified as putative allo-immunized and (2) the ones showing a lower MLR as putative tolerized responses. Analysis of the CD4+ T cells from the receptive partner stimulated by the partner's monocytes, compared with those of 3rd party cells showed a significantly higher CD4+ T cell proliferation stimulated by the partners' than 3rd party cells (p = 0.003). Similar results were found with the insertive partners' cells stimulated with the 3rd party CD4+ T cells (p<0.0001). As there was no significant difference in the MLR between the receptive and insertive subjects (p = 0.122) we combined them (mean 13.9±sem 2.0%) and analysed against the 3rd party cells (5.0±1.2%). A significant increase in CD4+ T cell proliferation was found in the immunized subjects  than that found with the partners' PBMC (1.4±1.4%), but the 5% level of significance was not reached, which was probably due to the small number of subjects (+ T cells was significant (p<0.0001). We have analyzed the MLR against the number of episodes of unprotected sexual intercourse over the past year by plotting each subject with reference to the allo-immune or tolerance response. None of the 3 cohorts showed any correlation between these parameters. We have similarly plotted the time of last intercourse against MLR, which also failed to show any correlation.Allogeneic stimulation t 1 year . These wsubjects . However+ T cells, which showed the combined CD8+ T cell proliferative response (partners A and B) between the immunized partners was significantly greater (31.0±4.7%) than the corresponding responses to the 3rd party PBMC  (+ T cell proliferative responses (17.7±5.6%) than those elicited by the 3rd party cells , compared with tolerized CD8+ T cells (17.7±5.6%) failed to reach the 5% level of significance (p = 0.088). Altogether the results suggest that homosexual intercourse may elicit mostly immune CD4+ T cell proliferative responses (17 out of 20 partners), compared with 9 out of 20 yielding CD8+ T cell proliferation. A decrease in CD4+ T cell proliferation, consistent with tolerization was found only in 3 out of 20 partners, but was greater with CD8+ T cells .A similar analysis was pursued with the CD8= 0.001) . Conversrd party cells  (+ T cell proliferation was found in the proportion responding to partners' cells (2.1±1.1), compared with those to the 3rd party irradiated cells (17.6±3.4) in the putative tolerized cells , as was also noted in the homosexual cohort. A significant increase (p<0.0001) was found in CD8+ T cell proliferation stimulated by the partners' cells (13.9±2.0%) compared with 3rd party cells (7.1±1.6%) in the immunized couples and the converse was found in the tolerized couples  in the allo-immunized (12.5±1.7%) compared with the tolerized couples (2.1±1.1%), this was not found with the CD8+ T cells  than those of 3= 0.001) . Convers+ T cell proliferative response (data not presented), when stimulation with the partners' monocytes were compared with those of 3rd party cells  were recruited practising condom protected sexual intercourse. Of the 16 subjects only 5 were immunized (p = 0.045), 4 tolerized (p = 0.036) and 8 showed no change in the CD4ty cells . The cor+FoxP3+ regulatory CD4+ T cells were identified by triple immunofluorescence flow cytometry with mAb to CD4, CD25 and FoxP3 antigens. A significant increase in the T regulatory cells (p = 0.013) was found in the cells from homosexual subjects, practising unprotected sex when stimulated with the partners' cells (6.6±0.9%), compared with the 3rd party cells , this was not significant, possibly due to the small number (n = 3) of tolerized subjects (data not presented). Immunization was significantly more frequent in the homosexual (14/16) than hetero-sexual cohorts , but no difference was found among the tolerized groups (p = 0.626). We have compared the T regulatory cells against the number of episodes of unprotected sexual intercourse for each subject in the 3 cohorts and found no correlation between these parameters.CD25ectively . Althoug+ T cell MLR and T regs was found in the immunized homosexual (p = 0.007)  were compared with CD8+ T cell MLR (31.0±4.7%), both for the homosexual (p = 0.001) and heterosexual cohorts . The tolerized cohorts, however, failed to show any of the above associations.A significant association between the CD4= 0.007)  and hete= 0.007) , suggest+ T cells within the PBMC population stimulated by the partners' cells compared with 3rd party cells in the homosexual  (rd party cells (p = 0.007). In heterosexual unprotected or protected subjects CD4+ T regs and IFN-γ producing cells failed to show a significant association  , though ociation  and 2.9,in vitro HIV-1 infectivity and the immunological parameters tested. No significant relationship was established with CD4+ T cells proliferation or with IFN-γ , heterosexual unprotected (p = 0.040) and protected (p = 0.030) cohorts , cultured up to 9 days and p24 was then assayed. Analysis of the heterosexual partners practising unprotected sex (9.5 median of 50% inhibition of virus) compared with those practising protected sex (145 median) showing a significant increase in virus dilution required for the PBMC to inhibit the virus  or heterosexual (p = 0.001 and p<0.0001) intercourse, respectively. However, whilst tolerization of CD4+ T cells was found only in 3/20 homosexual and 3/19 heterosexual partners, the CD8+ T cells were tolerized in 8/20 homosexual partners (p = 0.004) and in 6/20 heterosexual subjects (p<0.009). Altogether the CD8+ T cell responses were higher than those of CD4+ T cells, and CD8+ T cells were more likely to be tolerized than CD4+ T cells in both types of unprotected sexual activities. The mechanism governing these findings will need to be explored. As there was an HLA-class II mismatch of the DR and DQ alleles, which would generate MLR responses, sharing of HLA-class II would not account for the tolerance.The preliminary allogeneic studies + (p = 0.045) and CD8+ (p = 0.045) T cell proliferation, these were of considerably lower order of significance than those recorded in the unprotected cohorts . The proportion of CD4+ T cells in the allogeneic immunized protected partners was also smaller (5/16) than those in the unprotected partners (11/19). The tolerized subjects showed significant increase in CD4+ T cell proliferation (p = 0.036), unlike those in the unprotected cohort, but the tolerized CD8+ T cells showed comparable levels of significance in the 2 cohorts.Heterosexual subjects practising condom-protected sex were then analysed and although they also showed significant increase in CD4+CD25+ FoxP3+ regulatory T cells To interpret the effect of unprotected rectal and vaginal intercourse on allogeneic immunity and tolerance we studied CD4+ T cell proliferation. This was found both in CD4+ T cells by flow cytometry and testing CD4+ T cell culture supernatants by the Luminex bead technique (results not presented). IL-4 and IL-10 assays showed very low concentrations, suggesting that Th2 cells do not play a major role. Th17 cells showed very low levels (<1%), which were not amenable to analysis as reported by others, without enhancing IL-17 expression with cytokines, such as IL-1β and IL-6 + T reg cells were not studied, but CD4+ T regs may have inhibited CD8+ T cell proliferative responses, as the combined homosexual and heterosexual tolerized cohorts showed an inverse correlation between the two types of cells, though the 5% level of significance has not been reached .The overall aims were to study the 4 types of T helper cells; Th1 (IFN-γ), Th2 (IL-4 and IL-10), Th17 (IL-17) and T regulatory cells. Surprisingly IFN-γ showed no significant changes in the allogeneic immunized or tolerized homosexual or heterosexual partners and there was no significant association between IFN-γ and CD4Mucosal allogeneic immunization and tolerance has received limited attention, though the importance in mucosal infections, especially of the genital tract, is evident. Ejaculates contain CD4-positive T cells, macrophages, neutrophils, immature germ cells + T cells, 3/19 a decrease and 5/19 showed no change, the corresponding data for the protected partners were 4/16, 4/16 and 8/16, respectively. The higher proportion of allogeneic CD4+ T cell immunized and smaller number of unchanged MLR that were observed among the unprotected compared with protected heterosexual cohorts, is consistent with increased immune activation in those practising unprotected sex. However, this was not found in the CD8+ T cells. Furthermore, a larger proportion of CD4+ T reg cells was found in the allogeneic immunized homosexual partners (14/16–88%), compared with the heterosexual partners , both cohorts practising unprotected sex.This was not found in heterosexual partners practising protected sex.Overall it is noteworthy that unlike in heterosexual unprotected partners, in whom 11/19 showed evidence of significant increase in MLR of CD4Ex vivo HIV-1 infectivity studies suggest that allo-immunized heterosexual subjects practising unprotected sex may have acquired some protection to HIV-1 infection, as a significantly lower dilution of virus was inhibited by their PBMC, compared with those practicing protected sex (p = 0.028). A similar analysis of homosexual partners practising unprotected sex could not be pursued, as homosexual partners practising protected sex for a period over 1 year could not be recruited with any confidence. We then analysed any association between in vitro HIV-1 infectivity and the immunological parameters tested in the 3 cohorts. HIV-1 infectivity showed significant association with the T reg cells in all 3 cohorts (p<0.05 or p<0.01), but not with CD4+ T cell proliferation or IFN-γ production. As T reg cells were significantly associated with CD4+ T cell proliferation both in homosexual (p = 0.015) and heterosexual (p = 0.010) unprotected cohorts, the data support the concept that T reg cells play a regulatory role in allogeneic stimulation and HIV-1 infectivity of CD4+ T cells. We have not studied further the mechanism involved, but the T reg cells may inhibit CD4+ T cell activation and thereby infectivity of these cells. Indeed, there is evidence that allogeneic mediated T reg cells are efficient in suppressing transplantation responses Permission for this investigation was granted and approved by the Kings College Research Ethics Committee. Informed written consent was obtained from all participants. Recruitment was begun initially using a wide number of avenues, including mass-email requests through our host institutions of Guys and St Thomas' Hospitals and Kings College London, approaching asymptomatic patients seeking screening at sexually transmitted disease (STD) clinics and from members of the public who responded to advertisements. As no individuals from STD clinics met the criteria, we stopped using this avenue early on. Volunteers were excluded if they had a history of sexually transmitted or major infectious disease, at least a year prior to entry into the study. Patients were excluded if they were pregnant, or had been pregnant within 12 months of entry into the study. Patients were also excluded if they had any recent (within 12 months) severe illness, or if they had any chronic condition thought possibly to influence the study findings . This screening excluded patients who had blood transfusion within the past 12 months. We have not dealt in more depth with previous blood transfusions, as whole blood which may induce alloimmune responses has not been used in the UK over the past 11 years. As the mean age of our heterosexual “unprotected” cohort was 24.3±1.2 and the homosexual cohort 30.8±1.8, it is unlikely that a significant number would have had blood transfusions and of those that might have been transfused only about 2% develop alloimmune response. Subjects known or suspected to be HIV antibody positive were excluded and were only included if they were low risk for HIV infection or if they had a negative HIV antibody test within the previous 12 months. We enrolled three cohorts, all of which were monogamous, defined as partners that had no other sexual partners in terms of oral, anal or vaginal sex, whether protected or unprotected by means of barrier methods for the 12 months prior to enrollment in the study and for the duration of their participation in the study.Homosexual monogamous men practising unprotected sexual intercourse ; these were men having sex with men (MSM). The criteria for entry to this group were men having unprotected penetrative anal intercourse with the same male partner for at least the past 12 months. The partners in this group were only included if there was a clear history of them having had unprotected anal sex within this relationship, and no unprotected oral or anal sex with other partners within the previous 12 months. In addition, within this group, we recorded whether the individuals practised predominantly receptive (passive) intercourse, predominantly insertive (active), or a roughly equal mixture of both.Heterosexual monogamous partners practising unprotected sexual intercourse . The criteria for this group were that the couples were in a monogamous heterosexual relationship, otherwise the criteria were the same as for the MSM group.Heterosexual monogamous partners practising protected sexual intercourse . The criteria for this group were that the couples were in a monogamous heterosexual relationship, but that any act of sexual intercourse over the previous 12 months, including oral sexual intercourse, was practised using barrier contraception.The sexual history for all patients for all groups was taken by nurses experienced in sexual health and sexual history taking. Great care was taken, using open questioning, recording in private, and assurance of anonymity, in order to capture an accurate history. A detailed history of number, type and timings of intercourse were entered onto a structured written questionnaire for all subjects. This enabled data, such as the interval between the last intercourse and the time when the blood specimen was taken for this investigation to be evaluated.+ monocytes were enriched by depletion of CD14- cells using a Monocyte Isolation Kit . The purity of isolated monocytes was consistently greater than 90% when analyzed by flow cytometry with antibodies to CD14 (BD Biosciences). CD14+ monocytes were cultured in GM-CSF (1000 U/ml) and IL-4 (40 U/ml) supplemented medium for 4–5 days to differentiate into immature dendritic cells.Approximately 40 mls of blood was collected from each participant into EDTA filled tubes. Peripheral blood mononuclear cells (PBMC) were isolated by centrifugation of blood on a Ficoll-Hypaque density gradient . CD145/ml in supplemented RPMI culture medium, containing 200 mMol glutamine, 100 µg/ml penicillin and streptomycin and 10% FBS, from each partner were co-cultured in 96 well plates. One way MLR were set up with autologous, allogeneic or unrelated (third party) monocytes (stimulators), made up to 1×106/ml in supplemented RPMI culture medium. The cultures were incubated at 37°C for 7 days. At day 7 day supernatants from the cultures were harvested for cytokine analysis. Repeated tests with the cells from the same subjects stimulated with the partner's monocytes compared with 3rd party cells showed differences in percentages but the pattern of response remained the same with reference to 3rd party cells.To examine whether unprotected sex resulted in allo-immunization or allo-tolerization, CFSE labelled PBMCs (responders), made up to 2×10+CD4+FoxP3+ regulatory T cell analysis, unstained PBMCs (responder cells) were co cultured with autologous, allogeneic or unrelated (third party) monocytes (stimulators) in 96 well plates and incubated at 37°C for 7 days.For intracellular cytokine and CD255/ml in supplemented RPMI culture medium, were co-cultured in 96 well plates with 1×106/ml autologous or allogeneic immature DC. The cultures were incubated at 37°C for 7 days.For in vitro analysis of T cell proliferation to alloantigens, CFSE labelled PBMCs (responders), made up to 2×106 cells/ml. Aliquots of 2 µl/ml of 0.05 mM of CFSE (Invitrogen) were added to make a final working concentration of 1 uM and the cells were incubated for 10 minutes at 37°C. Staining was quenched by adding 5 volumes of ice cold RPMI to the cells, and then by incubation for 5 minutes on ice. The cells were then washed 3 times with RPMI and counted.PBMCs were suspended in pre-warmed PBS supplemented with 0.1% BSA at a final concentration of 1×10+ and CD8+T cells after 7 days of allostimulation, CFSE labelled cells were surface stained with antibodies to CD4 (APC-CY7) (BDBiosciences) and CD8 (APC) (BD Biosciences) at a 1∶100 dilution for 30 minutes at 4°C. After washing in PBS, cells were analysed by flow cytometry using a BD FACSCanto™ II, using the software DIVA.To determine the proliferative responses of both CD4To determine intracellular cytokine production, cultured cells were stimulated with 5 ng/ml PMA (Sigma), 0.5 µg/ml ionomycin (Sigma) and 0.7 µg/ml golgi stop (BD Biosciences) for 4 hours at 37°C. After washing with PBS, the cells were surface stained for 20 minutes at room temperature with 5 µl of an antibody to CD4 (APC) (BD biosciences) and 0.4 µg/ml of Live/Dead Fixable Near-IR (Invitrogen) to distinguish between dead and live cells. After washing twice with PBS, the cells were fixed with 100 µl/well IC fixation solution (eBioscience) and incubated for 20 minutes at room temperature. Cells were washed twice with 1x permeabilization buffer (eBiosciences). For intracellular staining, cells were incubated with 5 µl/well of IFN-γ (FITC) and 5 ul of IL-4 (PE) (BD Biosciences) for 20 minutes at room temperature. After washing twice in 1x permeabilization buffer cells were analysed by flow cytometry as described above.For cell surface staining, the cells were incubated with 5 µl of conjugated antibodies to CD4 (FITC) (BD Biosciences), CD25 (APC) (eBiosciences) for 30 minutes at 4°C.For intracellular staining for the regulatory cell marker FoxP3, cells were washed in cold PBS, and 1 ml of freshly prepared Fixation/Permeabilization buffer (eBiosciences) was added to the cells, which were then vortexed and incubated for 30 minutes at 4°C. After washing twice with 2 ml 1X permeabilization buffer, cells were blocked using 2% normal rat serum for 15 minutes at room temperature. After the blocking step, cells were incubated with 10 µl of either anti-human FoxP3 (PCH101) or rat IgG2a isotype control for 30 minutes at 4°C. After washing with 1 ml of 1X permeabilization buffer, cells were analysed by flow cytometry as described above.+ T cells (2×106/ml) were cultured with allogeneic or autologous immature DC (2×105/ml) for 7 days in 96 well plates at 37°C. For CD40L staining or isotype control staining , Golgistop  was added 10 hours before termination of the co culture. Cells were then stained and analysed by flow cytometry as described above. To differentiate between CD4+ Tregs and CD4+ effector T cells CD4+CD25+ T cells were gated according to the presence and absence of FoxP3 expression, respectively.CD46 cells were infected with serial dilutions of 1∶10−1 to 1∶10−4 multiplicity of HIV-1 strains for 3 h. The cells were washed three times with medium and cultured in triplicates at 1×105 per well in 96-well culture plates. Every 2 days, 100 µL of culture supernatant was replaced with 100 µL of medium supplemented with 20 IU interleukin 2. On day 8, the cell-free culture supernatant was assayed, with HIV-1 p24 Antigen ELISA . The infection of human cells with HIV-BaL strain was presented as the multiplicity of infection which inhibits 50% of the virus in PBMC.To test HIV-1 infection, PBMCs were activated with 10 µg/mL of PHA  in supplemented RPMI for 3 days then washed with medium and cultured in supplemented RPMI with 20 IU interleukin 2  overnight. Serial dilutions of primary HIV-1 strain BaL (CCR5-binding strain) were prepared with a starting concentration of 20 ng/100 µl of p24. Aliquots of 0·6×105′-TCA TCA TCC TCC TGA CAA TCG- 3′ and the antisense primer 5′ –CCA GCC CCA AGT TGA CTA TC-3′. DNA was extracted from PBMC using DNA isolation kit  according to the manufacturer's instruction. The purified DNA was stored at −20°C. HLA class I and II typing for HLA-A, -B, -C, -DRB and –DQB1 was carried out by the PCR technique with sequence specific primers; the primer sequences used have been previously reported. rd party cells (data not presented).For parametric data the results were expressed as mean (±sem) and student's paired or that for two means was used. For non-parametric data the Mann-Whitney test was used. The Chi square test was applied for analysis of associations, and Fisher exact test for cells with small frequencies. Inter-group analyses between the 3 cohorts using Student's t test for two means mostly showed no significant differences as it was apparent that there were two populations – putative allogeneic and tolerogenic – that yielded opposite responses when stimulated with the partner's monocytes and compared with those of the 3Figure S1+ and CD8+ T cell proliferative data in an immunized and tolerized subject shown in Representative illustrations of CD4(0.03 MB PDF)Click here for additional data file.Figure S2+ and CD8+ T cell proliferative data in an immunized and tolerized subject shown in Representative illustration of CD4(0.03 MB PDF)Click here for additional data file.


Sphingosine-1-phosphate (S1P) is a bioactive lipid that regulates a multitude of cellular functions, including cell proliferation, survival, migration and angiogenesis. S1P mediates its effects either by signaling through G protein-coupled receptors (GPCRs) or through an intracellular mode of action. In this study, we have investigated the mechanism behind S1P-induced survival signalling.2 receptors and Gi protein signaling. Our study also demonstrates that extracellular S1P stimulates cells to rapidly produce and secrete additional S1P, which can further amplify the NF-κB activation.We found that S1P protected cells from FasL-induced cell death in an NF-κB dependent manner. NF-κB was activated by extracellular S1P via S1PWe propose a self-amplifying loop of autocrine S1P with capacity to enhance cell survival. The mechanism provides increased understanding of the multifaceted roles of S1P in regulating cell fate during normal development and carcinogenesis. U73122 and Pertussis toxin were purchased from Sigma . VPC 23019 was from Avanti . FLAG-tagged TRAIL and SuperFasLigand were from Alexis . TRAIL was crosslinked with M2 anti-FLAG antibody  prior to stimulating cells. The S1P2,4,5 antibodies were from Santa Cruz Biotechnology , and the S1P1,3 antibodies were from both Santa Cruz and Cayman Chemicals . Pre designad SMARTpool siRNA's were purchased from Dharmacon .Fluo-3 AM and BAPTA AM were purchased from Molecular Probes . D-2 and 95% air. Cells were cultured in medium with serum replaced by 0.2% fatty acid free BSA 24 h prior to experiments. WM35 cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 5 μg/ml insulin, and 50 U/ml penicillin and 50 μg/ml streptomycin at 37°C in a water-saturated atmosphere of 5% CO2 and 95% air.HeLa cells and MEL-7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 50 U/ml penicillin and 50 μg/ml streptomycin at 37°C in a water-saturated atmosphere of 5% CO1, sense GGCTGGAACTGCATCAGTGCG, antisense GAGCAGCGCCACATTCTCAGAGC, 60°C, 223 bp; S1P2, sense CCGAAACAGCAAGTTCCACT, antisense CCAGGAGGCTGAAGACAGAG, 61°C, 197 bp; S1P3, sense AAGGCTCAGTGGTTCATCGT, antisense GCTATTGTTGCTGCTGCTTG, 61°C, 201 bp; S1P4, sense CCTTCAGCCTGCTCTTCACT, antisense AAGAGGATGTAGCGCTTGGA, 64°C, 223 bp; S1P5, sense AGGACTTCGCTTTTGCTCTG, antisense TCTAGAATCCACGGGGTCTG, 59°C, 201 bp.RNA was isolated using High Pure RNA Isolation Kit from Roche . For synthesis of cDNA SuperScript III from Invitrogen  was used and DynaZyme EXT from FinnZymes  was used for the PCR-reactions. All steps were done according to the instructions given by the manufacturers. PCR reactions were performed by first heating the reaction mix to 94°C for 5 minutes. This was followed by 29 cycles of 30 seconds at 94°C, 60 seconds at the annealing temperature and elongation 60 seconds at 72°C. The primers, annealing temperatures, and product lengths were: S1P3H] sphingosine  with fatty acid free BSA as carrier. Lipids were extracted by aspirating the culture medium and adding 500 μl of ice-cold methanol to the cells. Cells were scraped from the petri dishes and transferred to eppendorf tubes. The tubes were sonicated for 5 minutes and then centrifuged at 6,000 g for 10 minutes to remove cell debris. The supernatant was then transferred to glass vials. S1P was added to each sample for identification and the supernatant was evaporated. For measurements of secreted S1P, [3H]S1P was extracted from the medium as previously described previously [3H]S1P was counted using liquid scintillation. From a typical experiment the recovered counts of intracellular and secreted [3H]S1P at basal conditions were 449 ± 69 cpm and 175 ± 20 cpm, respectively in HeLa cells. Under similar conditions 225 ± 34 cpm was extracted from MEL-7 cells and 557 ± 61 cpm from the medium. WM35 cells had a basal cellular [3H]S1P of 213 ± 18 cpm and secreted 288 ± 55 cpm.Cells (roughly 270 000 cells per 35-mm cell culture plate) were incubated over night in medium with serum replaced by 0.2% fatty acid-free BSA. Cells were then stimulated with agonist or vehicle together with [eviously . BrieflyMluI and 3' SalI sites and cloned into the WPT-GFP lentiviral vector which had been digested with MluI and SalI to remove the GFP gene. Lentiviral vectors expressing the SphK-FLAG construct were produced by transient three plasmid cotransfection into HEK 293T cells by using calcium phosphate precipitation. The three plasmid mixture consisted of 14.5 μg WPT-SphKFLAG, 8.3 μg pCMVΔR8.91 and 2.1 μg MD.G . The virus-containing media were harvested 48 hours later by filtering the media through 0.45 μm pore size filter and centrifuging at 16 000 g for 2.5 h at +4°C. The resulting pellets were resuspended in 200 μl serum free DMEM. For transduction HeLa cells were plated on 6-well plates (1 × 105 per well) and 24 hours later virus together with 8 μg/ml Polybrene was added at multiplicity of infection 10 and incubated for 6 hours after which time the medium was replaced with fresh medium.Human SphK cDNA was cloned and FLAG -tagged at the 3' end according to Pitson et al . The SphThe cells were grown to 90% confluency, and transfection was done with N-TER transfection reagent according to the manufacturer's protocol for serum-free transfection with slight modifications. The siRNA was added to the cells at a final concentration of 100 nM. Following a 24 hour incubation with the siRNA reagent, the medium was changed to fresh medium containing 0.2% Fatty acid free BSA. Following another 24 h incubation the cells were used for experiments.2, 0.2 mM EDTA, 20 mM Hepes (pH 7.9), 0.5 mM DTT, and 0.5 mM PMSF. The extract was then centrifuged at 15,000 × g for 20 minutes at +4°C. The supernatant was collected and protein concentrations were determined. Two methods for assaying NF-κB (p65) DNA-binding activity were used. NFκB transactivation capacity was measured from the extracts either by using a Trans-AM NF-κB (p65) transcription factor assay kit  according to the manufacturers' instructions, or by electrophoretic mobility shift assay (EMSA). The results from the Trans-AM NF-κB (p65) transcription factor assay kit are presented as percent activation with the stimulated control set to 100% and the unstimulated control as 0%. This was made to compensate for between-experiment differences in NF-κB activation. For the EMSA experiments, the consensus NF-κB binding site (5'-AGCTTCAGAGGGGACTTTCCGAGAGG-3') was 32P-labeled. Protein-DNA complexes were separated on a native 4% polyacrylamide gel. The gel was dried and exposed to autoradiography film over night. Both methods measure NF-κB activity by detecting it binding to its consensus binding sequence.Cells grown on 60 mm petri dishes were harvested and pelleted in ice cold PBS. The cell pellet was quick-freezed and resuspended in 150 μl buffer containing 25% glycerol, 0.42 M NaCl, 1.5 mM MgClCells were assayed using an active caspase-3 detection kit from BD Pharmingen according to the manufacturers' instructions. In brief, cells were collected in eppendorf tubes, spun down and washed twice in 1 ml PBS. The cells were resuspended in 150 μl Cytofix/Cytoperm™ Solution. Following a 20 minute incubation on ice the cells were washed twice with 0.5 ml Perm/Wash™ Buffer. The cells were then incubated with the phycoerythrin-labelled antibody against active caspase-3 (1:20 in 100 μl Perm/Wash™ Buffer). The cells were washed once in 1 ml Perm/Wash™ Buffer. The cells were resuspended in 0.5 ml Perm/Wash™ Buffer and were then analyzed by flowcytometry using a BD FACScan and Cell Quest software.L from Santa Cruz  and anti-Hsc70 from Stressgen . HRP conjugated secondary antibodies were used, and bands were exposed on film by chemiluminescence.35 mm-petri dishes were washed once with cold HBSS, and scraped in 70 μl lysis buffer (10 mM Tris/HCl (pH 7.7), 150 mM NaCl, 7 mM EDTA, 0.5% NP-40, 0.2 mM PMSF, and 0.5 μg/ml leupeptin). Lysates were kept on ice for 15 minutes and were then centrifuged at 10,000 g for 15 minutes. 3 × Laemmli's buffer was mixed with the supernatant and the solution was heated to 95°C for 3 minutes. Proteins were separated by 10% SDS-PAGE and transferred onto a nitrocellulose membrane. The primary antibodies used were anti-Bcl-xt test for paired observations. When three or more means were tested, one way ANOVA was performed followed by Dunnett's test for multiple comparisons against a single control. Statistical significance (p < 0.05) is denoted with *.Results are expressed as means ± SEM from a minimum of three independent experiments. Statistical analysis was made using Student's It has been firmly established that S1P is a cytoprotective agent. However, the downstream mechanisms are pleiotropic and have only been partially identified. To study the S1P-induced survival signaling pathways we first needed to identify a reproducible and quantifiable endpoint. NF-κB is a well-defined survival factor, and there are reports showing that S1P activates NF-κB by signaling through G-protein coupled S1P receptors -33. NF-κ1-3 and S1P5 were detectable by PCR and Western blot in HeLa cells  inhibitor PD98059 was without an effect , mitogen activated protein kinase (MAPK) and Cat Figure . The S1P, Figure . These rectively ,20. Thesper se did not render the tumor cells apoptotic, but sensitized them to apoptosis induced by TRAIL -4-(p-chlorophenyl)thiazole.S1P: sphingosine-1-phosphate; NFκB: nuclear factor κB; SphK: Sphingosine kinase; DMS: N,N-dimethylsphingosine; SKi: 2-, the Sigrid Juselius Foundation , The Liv och Hälsa Foundation (KT), Svenska Kulturfonden (KT) and the Academy of Finland . The study was done as a part of the Receptor Structure and Function program at the University of Turku, Åbo Akademi University and the National Public Health Institute, and as a part of the Center of Excellence in Cell stress consortium at Åbo Akademi University. Tomas Blom, Annika Meinander and Nina Bergelin were supported by the Turku Graduate School of Biomedical Sciences during this work. The funding bodies had no role in in study design, data collection, analysis, interpretation, in writing the manuscript, or in the decision to submit the paper for publication.


N-glycosylated. In addition IgG3 appeared to be O-glycosylated and could bind the lectin jacalin. Staphylococcal protein A displayed weak binding for the equine IgGs in the order: IgG1 > IgG3 > IgG4 > IgG7 > IgG2 = IgG5 > IgG6. Streptococcal protein G bound strongly to IgG1, IgG4 and IgG7, moderately to IgG3, weakly to IgG2 and IgG6, and not at all to IgG5. Analysis of antibody effector functions revealed that IgG1, IgG3, IgG4, IgG5 and IgG7, but not IgG2 and IgG6, were able to elicit a strong respiratory burst from equine peripheral blood leukocytes, predicting that the former five IgG subclasses are able to interact with Fc receptors on effector cells. IgG1, IgG3, IgG4 and IgG7, but not IgG2, IgG5 and IgG6, were able to bind complement C1q and activate complement via the classical pathway. The differential effector function capabilities of the subclasses suggest that, for maximum efficacy, equine vaccine strategies should seek to elicit antibody responses of the IgG1, IgG3, IgG4, and IgG7 subclasses.Recombinant versions of the seven equine IgG subclasses were expressed in CHO cells. All assembled into intact immunoglobulins stabilised by disulphide bridges, although, reminiscent of human IgG4, a small proportion of equine IgG4 and IgG7 were held together by non-covalent bonds alone. All seven IgGs were  The hor in vivo . Early s in vivo . Of the  in vivo .Streptococcus equi  within the cloning vector pBluescript II . For each subclass, the 5′ UTR and γ constant region cassette was then subcloned into pcDNA3.1VNip downstream of the mouse VH gene.The mouse Vn vector  into pcDescribed . For con2.2CHO-K1 cells, which had previously been stably transfected with the mouse λ L chain specific for NIP, were transfected with one of the γ1–γ7 H chain expression vectors, as previously described . Superna2.32L2) were pooled for further analysis. The antibodies were analysed by SDS-PAGE and Western blotting on 8.4% acrylamide gels under reducing and non-reducing conditions. Gels were stained with Coomassie brilliant blue and Western blots were probed with HRP-conjugated rabbit anti-mouse λ L chain (0.2 μg/ml) or anti-horse IgG antibodies. Horse serum IgG  and recombinant human IgG1 , , and mouse monoclonal antibodies (mAb) against horse IgGa (CVS48), IgGb (CVS39), IgGc (CVS53) and IgG(T) (CVS38) (kindly provided by Serotec). Secondary antibody used to detect binding of the mAb was a goat anti-mouse IgG1-HRP conjugate diluted 1/10,000 (kindly provided by Serotec). In addition, HRP-conjugated goat polyclonal anti-horse IgG H + L chain (kindly provided by Bethyl Laboratories) and goat anti-horse IgG Fc (Rockland) were tested in ELISA. Antigen-capture ELISAs were performed as above using 250 ng/ml of each purified reqIgG subclass. Detection antibodies were diluted 1/10,000  or 1/100 (anti-IgGa mAb), 1/200 (anti-IgGb mAb), 1/10 (anti-IgGc mAb) and 1/500 (anti-IgG(T) mAb). The goat polyclonal anti-horse IgGa, IgGb, IgGc and IgG(T) antibodies were also tested in immunoblotting. Purified reqIgGs (1 μg) were electrophoresed under reducing and non-reducing conditions and the detection antibodies were diluted as for ELISA.2.5N-linked sugars was carried out using a GlycoPro enzymatic deglycosylation kit  according to the manufacturer's instructions. Following transfer to nitrocellulose, membranes were probed with the appropriate HRP-conjugated goat anti-horse IgG subclass antibody or either Con A-biotin  or jacalin-biotin (diluted 1/5000)  followed by streptavidin-AP . Recombinant human IgG1 and IgA1 (ReqIgGs (1 μg) were electrophoresed on 8.4% acrylamide gels under reducing conditions alone or following reduction and de-glycosylation. Removal of and IgA1  were use2.6Interaction of reqIgGs with protein A and protein G was analysed by ELISA. NIP-BSA coated wells were incubated with 100 μl (0.25 μg/ml) of one of the purified reqIgGs or recombinant human IgG1 for comparison. Binding to protein A or protein G was detected by incubation with 100 μl of serial dilutions of either HRP-conjugated protein A (0–50 μg/ml in PBS-T) or HRP-conjugated protein G (0–5 μg/ml in PBS-T) . Plates were developed with ABTS and absorbance read at 405 nm.2.72, 2 mM CaCl2, 0.05% Tween-20, 0.1% gelatin and 0.5% BSA for 1 h at room temperature. After washing, plates were incubated with either a 1/800 dilution of sheep anti-C1q-HRP (Serotec) or a 1/500 dilution of biotin-conjugated anti-C5b-9 , followed by streptavidin-HRP (Dako) diluted 1/1000 in PBS-T, 0.5% BSA for 1 h at room temperature. Plates were developed as above.Chemiluminescence assays were carried out as previously described  using 1033.1Transfection of each of the equine γ1–γ7 H chain expression vectors into CHO cells stably expressing a compatible mouse λ light (L) chain allowed expression of all seven subclasses. Expressed Ig was purified by antigen affinity chromatography followed by FPLC. All seven IgGs eluted from FPLC as a single major peak representing monomer. Minor peaks eluting earlier were attributed to antibody that had aggregated during purification. For each subclass, fractions corresponding to the major peak were pooled and used for all subsequent experiments.2), suggesting that a small proportion of the IgG4 and IgG7 molecules lack inter-H and/or H–L disulphide bonds and are stabilized by non-covalent interactions alone. Hence, under the native conditions of FPLC only a single major peak of fully assembled IgG was seen.SDS-PAGE analysis of FPLC purified reqIgGs confirmed that the reqIgs assembled into intact Igs stabilised by interchain disulphide bridges A. Under 3.2Currently available anti-horse IgG antibodies are categorised on the basis of reactivity against horse IgG subclasses defined under the previous nomenclature of IgGa, IgGb, IgGc, and IgG(T). We sought to analyse the reactivity of these antibodies with the seven reqIgG subclasses. The results, obtained by ELISA, are summarised in The polyclonal anti-horse IgG (H + L) and anti-horse IgG Fc antibodies recognised all seven IgG subclasses but gave varying signal strengths in the ELISA see . Anti-hoThe reactivities of the anti-horse polyclonal Abs with the horse IgG subclasses when assessed by immunoblotting under non-reducing conditions A mirrore2) and half molecules (HL), suggesting, again, an absence of inter-H and/or H–L disulphides in small fractions of these two subclasses.Under non-reducing conditions, immunoblotting with goat anti-IgGb A reveale3.3N-glycosylation site within the CH2 domain. Additional potential sites are found in the CH1 domain of IgG3 and IgG5 and in the CH2 domain of IgG2. To determine whether N-glycans were attached to any of these sites, the reqIgGs were probed under reducing conditions with concanavalin A (Con A), which is specific for N-linked glycans. Con A bound to all reqIgG subclasses confirming that the IgGs are indeed N-glycosylated ) was lost following the removal of N-glycans, suggesting that the epitopes recognized by this anti-horse reagent are in some way glycan-dependent. The IgG5 H chain migrated as two discrete bands following treatment with N-glycanase, possibly reflecting partial removal of the two potential N-linked glycans in a proportion of the molecules treated.Treatment of reqIgGs with H chains , confirm3.4Binding of protein A to reqIgGs was generally much weaker than the binding of protein G , in keepProtein A showed the highest level of binding to IgG1 and relatively low levels of binding to the other subclasses in the order: IgG3 > IgG4 > IgG7 > IgG5 = IgG2 > IgG6. Protein G showed strong binding to IgG4 and IgG7, closely followed by IgG1. IgG3 displayed intermediate binding, while binding to IgG6 and IgG2 was low, and IgG5 did not bind. 3.5We studied FcγR-mediated cellular responses by assessing the capacity of the antibodies to trigger a respiratory burst in equine peripheral blood leukocytes (PBL). Recombinant IgG1, IgG4, IgG5 and IgG7, and to a lesser extent IgG3, all triggered strong respiratory bursts, whereas IgG2 and IgG6 elicited only very weak responses .Binding of C1q and activation of the classical complement pathway by the reqIgGs was assessed using ELISA. Human serum was used as a source of complement due to the ready availability of antibodies to detect human C1q and human C5-9 complex. The C1q–IgG interaction is highly conserved throughout evolution and C1q is known to react with IgG from different species . The abi42L2) molecules with the expected relative molecular weight of approximately 150 kDa. Hence, in terms of structure, they appear to be representative of natural horse IgGs.Our studies show that the CHO expression system previously described for human antibodies  can be uO-linked as well as N-linked glycans attached to the H chain. O-glycosylated forms of rabbit IgG and mouse IgG2b in which the O-glycans are attached to the hinge region have been described , half molecules (HL) and L chain dimers (L2) under denaturing conditions, suggesting that a small proportion of the molecules lack H–L or H–H disulphide bonds, akin to human IgA2m(1) and human IgG4, respectively. The human IgG2, IgG3 and IgG4 subclasses are disulphide bonded to the L chain through Cys131 in the CH1 domain. The equivalent Cys is missing in equine IgG4 and IgG7, and may account for the tendency for a minor proportion of these subclasses to exist in forms lacking H–L bonds. The majority of equine IgG4 and IgG7 molecules are present at molecular weights corresponding to intact (H2L2) antibodies, suggesting that H–L disulphide bonds can exist. Human IgG1, which lacks Cys131 in the CH1 domain, instead forms a disulphide bond to the L chain through Cys220 in the upper hinge    play a c-Ser239) . In the -Ser239) . Thus, aActivation of the classical complement pathway by IgG may be initiated through the binding of the first complement component C1q, to two or more adjacent IgG molecules. Human IgG subclasses bind C1q in the rank order IgG3 > IgG1 > IgG2 while IgG4 shows no significant binding . Early sThe C1q binding site lies within the CH2 domain of IgG, but appears not to be conserved precisely across species. Glu318, Lys320 and Lys322 are necessary for C1q binding by mouse IgG2b, whilst in human IgG1 or IgG3 Asp270, Lys322, Pro329 and Pro331 appear important with minimal contribution from Glu318 and Lys320 . A compaOur data on the effector function capabilities of the IgG subclasses have important new implications for the design of effective horse vaccines. It is clear that to achieve maximal protection via FcγR- and complement-mediated elimination mechanisms, vaccines should seek to elicit IgG antibodies of the IgG1, IgG3, IgG4 and IgG7 subclasses. Vaccines that trigger only IgG2, IgG5 or IgG6 antibodies are predicted to offer less effective protection. Since IgG plays key roles in both serum and mucosal compartments in the horse, these considerations are applicable to both systemic and mucosal vaccination strategies.Streptococcus equi  contribute to protection against equine influenza virus , Streptocus equi  and Rhodcus equi . Indeed,cus equi , whereascus equi . The curcus equi  and acutcus equi  is complrophages . DrawingInternational meetings have highlighted the need for further research into the functional roles of equine IgG subclasses and for the development of tools to study equine Igs . The req


Reelin is a large secreted protein of the extracellular matrix that has been proposed to participate to the etiology of schizophrenia. During development, reelin is crucial for the correct cytoarchitecture of laminated brain structures and is produced by a subset of neurons named Cajal-Retzius. After birth, most of these cells degenerate and reelin expression persists in postnatal and adult brain. The phenotype of neurons that bind secreted reelin and whether the continuous secretion of reelin is required for physiological functions at postnatal stages remain unknown.Combining immunocytochemical and pharmacological approaches, we first report that two distinct patterns of reelin expression are present in cultured hippocampal neurons. We show that in hippocampal cultures, reelin is secreted by GABAergic neurons displaying an intense reelin immunoreactivity (IR). We demonstrate that secreted reelin binds to receptors of the lipoprotein family on neurons with a punctate reelin IR. Secondly, using calcium imaging techniques, we examined the physiological consequences of reelin secretion blockade. Blocking protein secretion rapidly and reversibly changes the subunit composition of N-methyl-D-aspartate glutamate receptors (NMDARs) to a predominance of NR2B-containing NMDARs. Addition of recombinant or endogenously secreted reelin rescues the effects of protein secretion blockade and reverts the fraction of NR2B-containing NMDARs to control levels. Therefore, the continuous secretion of reelin is necessary to control the subunit composition of NMDARs in hippocampal neurons.Our data show that the heterogeneity of reelin immunoreactivity correlates with distinct functional populations: neurons synthesizing and secreting reelin and/or neurons binding reelin. Furthermore, we show that continuous reelin secretion is a strict requirement to maintain the composition of NMDARs. We propose that reelin is a trans-neuronal messenger secreted by GABAergic neurons that regulates NMDARs homeostasis in postnatal hippocampus. Defects in reelin secretion could play a major role in the development of neuropsychiatric disorders, particularly those associated with deregulation of NMDARs such as schizophrenia. Reelin is a large secreted protein of the extracellular matrix 2+, ionotropic glutamate receptors activity In the central nervous system, the spatiotemporal expression of reelin varies markedly from the prenatal to adult stage. Although the vast majority of Cajal-Retzius cells which express reelin during embryonic development Reelin serves different functions during brain development and adulthood. During embryonic development, reelin is crucial to the correct cytoarchitecture of laminated structures Here, we demonstrate that in hippocampal cultures, reelin is synthesized and secreted by GABAergic neurons. We show that continuous reelin secretion is necessary to maintain the subunit composition of NMDARs. These data are consistent with the hypothesis that reelin is a transneuronal messenger that regulates glutamate receptor homeostasis in post-natal hippocampal neurons.We analyzed, during maturation, the patterns of reelin expression in cultured hippocampal neurons with the G10 antibody  = 16.7, P<0.001; The expression profiles of these two types of IR varied similarly between 6 and 14 div . The proAlthough, these two types of labelling have been reported in vivo in different brain areas including the hippocampus of adult rodents  = 33.05, P<0.001). The BFA-dependent blockade was fully reversible following drug washout: the fraction of neurons with punctate reelin IR following BFA recovery was not different from the value obtained prior treatment , an inhibitor of protein translocation from the endoplasmic reticulum to the Golgi apparatus We next examined whether neurons secreting reelin were associated with a selective neuronal subtype. Double labeling of reelin with GAD67 or GAD65, two different isoforms of glutamate decarboxylase, showed that virtually all intense reelin IR were GAD67- or GAD65-positive . To dete = 12.0, P<0.01) and was abolished after 9 and 16 hours . The vehicle had no effect on either reelin-positive populations , a subclass of the ionotropic excitatory L-glutamate receptors, are heteromeric channels of obligatory NR1 subunits in combination with either NR2 (NR2A-NR2D) or NR3 subunits 2+). NMDARs activation was monitored by the increase of intracellular Ca2+ concentration upon NMDA application using the fluorescent Ca2+ indicator, Oregon Green BAPTA1-AM. The sensitivity of NMDA-stimulated Ca2+ influx to the antagonist ifenprodil was used to quantify the fraction of NR2B-containing NMDARs . One might expect treatment with cycloheximide to diminish the punctate reelin IR population by limiting the pool of synthesized reelin available for binding. Several explanations can account for the lack of effect within the time scale of the experiment; the turnover rate of unbound reelin could be very slow or alternatively, the pool of secretable reelin may not be sufficiently depleted and and thus still be readily releasable even after synthesis have been blocked for a long period of time. Finally, our observation is compatible with reelin recycling in punctate reelin IR neurons since it is known that after binding to lipoprotein receptors, reelin undergoes endocytosis The present data and our previous work Several studies have shown that, in the postnatal and adult brain, reelin modulates neuronal plasticity, including long-term potentiation, dendritic branching, maturation and surface diffusion of NMDARs Several molecular mechanisms could account for the effects of reelin secretion. First, the activation of the reelin signaling pathway could result in the phosphorylation of NMDARs thus upregulating NR2B-dependent responses Based on our data, we propose that defects in reelin secretion could play a major role in the development of neuropsychiatric disorders. Particularly those associated with deregulation of NMDARs homeostasis and lower expression levels of reelin, such as schizophrenia 2. Neurons were grown in Minimum Essential Medium  supplemented with 5% of Serum Supreme (Biowhittaker), 28.3 mM D-glucose and 0.1% of Mito+ Serum Extender (Becton Dickinson). Media were fully renewed the day after plating and exchanged weekly. Cultures were kept in a humidified atmosphere of 5%CO2-95%O2 at 36.5°C. To avoid glial cell multiplication, cultures were treated at 9 day in vitro (div) with 0.2 mg/ml 5′-fluoro-2-deoxyuridine and 0.5 mg/ml uridine .Hippocampi were dissected from postnatal day 0 Swiss mice brains. First, tissues were enzymatically dissociated for 40 min at 37°C in HBSS containing papain . Papain was then inactivated with Trypsin Inhibitor  in the presence of bovine serum albumin . Hippocampi were then mechanically triturated in the culture medium. Finally, dissociated cells were plated on poly-L-lysine coated round coverslips  at a density of 462 per mmAll experiments were conducted in strict compliance with European directives and French laws on animal experimentation (authorization number 3307016).Brefeldin A  was used as previously described (0.75 µg/ml) Cultures were treated at 9 div for 24 hours with different concentrations - 25, 40 and 50 µg/ml - of either the Receptor-Associated Protein fused with glutathione S-transferase (GST-RAP) or the control GST Cycloheximide (Sigma-Aldrich) treatments were performed at 13 div. Cycloheximide was added directly to the culture medium at a final concentration of 10 µM. Absolute ethanol with the same final volume percentage was used as a control.Hippocampal cultures were fixed in 4% paraformaldehyde-containing Phosphate Buffer Solution (PBS), pH 7.4 for 40 min at room temperature. Quenching was performed with 0.1 M glycine (Sigma-Aldrich) in PBS for 45 min. Permeabilization was achieved with PBS containing 0.1% triton X-100 (Sigma-Aldrich) for 5 min or PBS containing 0.05% saponin for 30 min. Non specific binding was blocked with 2 sequential incubations of 15 min duration with blocking buffer. Blocking buffer consisted of PBS containing either 10% donkey serum (Chemicon) or 0.2% Bovin Serum Albumin (Sigma-Aldrich) plus 0.05% saponin. Bovin Serum Albumin was used at 1% with anti-ApoER2 and anti-VLDLR primary antibodies. Cells were incubated overnight at 4°C in a humidified atmosphere with the primary antibodies diluted in blocking buffer. Preparations were then washed twice 15 min followed by a 30 min rinse in blocking buffer and incubated with the secondary antibodies for 80 min at room temperature in a humidified atmosphere. Cells were then rinsed extensively with the blocking buffer and mounted on glass coverslips in Aqua-PolyMount (PolySciences).Reelin was detected with the monoclonal antibody G10  and visualized with a cyanine 3 (Cy3)-conjugated anti-mouse IgG  or an Alexa Fluor 568-conjugated anti-mouse IgG . MAP2 was revealed with a rabbit polyclonal antibody (1∶500) and visualized with Alexa Fluor 647-conjugated anti-rabbit IgG . To visualize GABAergic neurons, we used antibodies directed against different isoforms of the GABA synthesizing enzymes, the rabbit anti-Glutamic Acid Decarboxylase 67  and the rabbit anti-GAD65 . These primary antibodies were revealed with cyanine 5 (Cy5)-conjugated anti-rabbit IgG  or Alexa Fluor 488-conjugated anti-rabbit IgG . The vesicular glutamate transporters 1 and 2 were detected with rabbit anti-VGlut1 (1∶1000) or VGlut2 (1∶2500) respectively and revealed with Cy5-conjugated anti-rabbit IgG . Reelin lipoprotein receptors were labeled with the rabbit anti-ApoER2  and the mouse anti-VLDLR  antibodies revealed with Alexa Fluor 488 anti-rabbit IgG  and Cy3-conjugated anti-mouse IgG  respectively.Slides were viewed with a 40× oil objective on an Axiophot II microscope . Image acquisition was done with a Quantix digital cooled CCD camera  driven by IPLab software . The exposure settings were kept identical for each condition. For each fluorescent image that was acquired, the corresponding phase-contrast field was collected. The 12 bit images were analysed with MetaMorph software 7.1.2.0  as follows. Somata were demarcated on the phase-contrast image, and the regions of interest (ROIs) were transferred automatically on the corresponding fluorescent images. Positive ROIs were selected using intensity thresholds determined by the distribution of intensities obtained amongst different experiments. Positive somata were then counted over all of the images acquired on coverslips from one experiment and normalized to the total number of neurons detected on the corresponding phase-contrast images.2+-free recording medium containing (in mM): NaCl (160), KCl (2.4), Hepes (10), glucose (10), glycine (0.02) and CaCl2 (3), pH = 7.3 and Osm = 325 mOsm/l. Coverslips were then mounted onto a custom made recording/perfusion chamber positioned on the movable stage of an inverted microscope Olympus IX-71. Cells were superfused (0.7 ml/min) with the recording medium. Signals were detected using a CCD camera (Quantem 512SC) and acquired with the Metamorph software 7.1.2.0 . Images were acquired with the filter set 41001, HQ 480/40  for excitation and HQ 535/50 (Chroma Technology) for emission. Recording sessions were between 40 and 45 minutes. The acquisition rate was 0,017 Hz during baseline (10–12 min) and ifenprodil application  and 0.5 Hz during NMDA (100 µM) and NMDA (100 µM)+ifenprodil (3 µM) applications. Phase contrast images were acquired prior and after each experiment to control for cellular integrity.Hippocampal neurons were loaded with 2 µM Oregon Green 488 BAPTA-1 AM (Molecular Probes) for 30 min at 37°C in culture medium. Coverslips were then washed 4 times with the MgData were analysed using the Metamorph software 7.1.2.0. After background correction, positive ROIs were demarcated only on somata whose integrity was confirmed in phase contrast images. Raw values (Ft) were normalized to the average of baseline calculated before NMDA application (Fr) and data were plotted as ΔF/F = (Ft−Fr)/Fr. NMDA responses were calculated as ΔF/F (NMDA±ifen)−ΔF/F (before NMDA±ifen).All experiments were performed at least in 3 different cultures, n is the number of cultures used for each condition unless stated otherwise. All data are expressed as the mean±SEM. Statistical analysis were performed by ANOVA followed by Tukey's multiple comparison test where significant .Figure S1Time course of reelin immunostaining during treatment with BFA or vehicle. (A) Representative images of intense and punctate reelin IR after 3 hours and 16 hours of BFA treatment and after BFA recovery. (B) Treatment with vehicle does not affect the percentage of reelin immunoreactive neurons at various time points. Values for intense reelin IR were: before treatment (0 hr) 21.1±1.2%, n = 8; 3 hours 19.7±2.0%, n = 3; 6 hours 21.2±1.0%, n = 3; 9 hours 18.8±1.3%, n = 5; 16 hours 21.2±0.4%, n = 6 and recovery 21.3±2.6%, n = 6. Values for punctate reelin IR were: untreated (0 hr) 18.0±1.4%, n = 8; 3 hours 17.7±2.5%, n = 3; 6 hours 19.0±1.6%, n = 3; 9 hours 18.1±1.4%, n = 5; 16 hours 17.4±1.1%, n = 6 and recovery 17.7±0.2%, n = 6.(6.53 MB TIF)Click here for additional data file.Figure S2Secretion blockade decreases the levels of total and full-length reelin present in the medium of cultured hippocampal neurons. (A) Example western blot of the time course of BFA treatment on reelin secreted in the medium of 13 div hippocampal cell cultures. Bands corresponding to full-length reelin (400 kDa) and proteolytic products (320 and 180 kDa) were disclosed by the G10 antibody. Reelin was not detected in medium not exposed to cultured neurons (medium).  Time course of the effect of BFA treatment on the levels of secreted reelin measured by western blot. For each condition, values are expressed as the percentage±sem of the corresponding vehicle control. **P<0.01, ***P<0.001. (B) Densitometry measurements of total reelin expressed as the sum of the densities of the 3 forms, full-length reelin (400 kDa) plus the two reelin fragments (320 and 180 kDa). A significant decrease is observed after 3 hours (53.8±2.9% of vehicle) and 9 hours (41.3±0.4% of vehicle) of treatment compared to 1 hour treatment with BFA (93.9±2.1% of vehicle) or vehicle  = 7.7, P<0.01). (C) Densitometry measurements of the 400 kDa band, corresponding to full-length reelin, after treatment with either vehicle, 1 hour BFA (84.8±3.0%), 3 hours BFA (42.8±4.0%) or 9 hours BFA  = 99.1, P<0.001).(5.90 MB TIF)Click here for additional data file.Figure S3Time course of reelin immunostaining during treatment with cycloheximide or vehicle. (A) Representative images showing intense and punctate reelin IR after 3, 9 and 16 hours of cycloheximide treatment (CHX). (B) The percentage of reelin immunoreactive neurons was not changed during incubation with vehicle at various time points. Values for intense reelin IR were: before treatment 23.7±1.7%, n = 4; 1 hour 21.7±1.7%, n = 3; 3 hours 19.1±2.7%, n = 3; 6 hours 18.7±2.9%, n = 4; 9 hours 20.2±3.1%, n = 3 and 16 hours 19.2±1.4%, n = 3. (C) Values for punctate reelin IR were: before treatment 18.4±0.8%, n = 4; 1 hour 18.2±2.6%, n = 3; 3 hours 17.9±2.4%, n = 3; 6 hours 19.7±3.1%, n = 4; 9 hours 17.6±1.0%, n = 3 and 16 hours 17.1±6.2%, n = 3.(8.10 MB TIF)Click here for additional data file.Figure S4Dual labelling of reelin and VLDLR. Representative image of a punctate reelin IR neuron (A) showing co-expression of VLDLR (B) in a 14 div hippocampal culture.(1.62 MB TIF)Click here for additional data file.Figure S5Subsequent applications of NMDA do not induce run-down of Ca2+ responses. Example of Ca2+ responses kinetics recorded in a 10 div neuron during subsequent applications of NMDA. The intensity of Ca2+ response remained stable during consecutive NMDA applications. On average the amplitude of the Ca2+ response obtained during the second application of NMDA represented 94.5±3.3% of the response evoked by the first application of NMDA (n = 33 neurons).(1.56 MB TIF)Click here for additional data file.Table S1Supplementary Table(0.03 MB DOC)Click here for additional data file.Text S1Supporting Materials(0.04 MB DOC)Click here for additional data file.


Bacillus subtilis. Earlier studies had indicated that CspB could maintain its folded structure in fibrils, when it was separated from the amyloidogenic segment by a long linker. When CspB is directly fused with the amyloidogenic segment, it unfolds because its N-terminal chain region becomes integrated into the fibrillar core, as shown by protease mapping experiments. Spacers of either 3 or 16 residues between CspB and the amyloidogenic segment were not sufficient to prevent this loss of CspB structure. Since the low thermodynamic stability of CspB (ΔGD = 12.4 kJ/mol) might be responsible for unfolding and integration of CspB into fibrils, fusions with a CspB mutant with enhanced thermodynamic stability (ΔGD = 26.9 kJ/mol) were studied. This strongly stabilized CspB remained folded and prevented fibril formation in all fusions. Our data show that the conformational stability of a linked, independently structured protein domain can control fibril formation.Conversion of native proteins into amyloid fibrils is irreversible and therefore it is difficult to study the interdependence of conformational stability and fibrillation by thermodynamic analyses. Here we approached this problem by fusing amyloidogenic poly-alanine segments derived from the N-terminal domain of the nuclear poly (A) binding protein PABPN1 with a well studied, reversibly unfolding protein, CspB from  Congenital protein misfolding diseases moved into the focus of intensive investigation during the past decades  is histochemically characterized by amyloid-like intranuclear inclusions consisting mainly of the nuclear RNA binding protein PABPN1 Bacillus subtilisThe recombinantly produced wild type form of the N-terminal domain of PABPN1 with ten Ala residues forms fibrils very slowly In this study, we explored how the folded state of CspB in the fibrils depends on the length of the linker between the amyloidogenic oligo-Ala segment and CspB. In a reciprocal approach, we investigated how fibril formation is affected when the amyloidogenic segment is linked with a thermodynamically stabilized variant of CspB PABPN1 using the QuickChange II Site-Directed Mutagenesis Kit (Stratagene) and the following primers: 5′-GCA GGA GCA GCA GGA GGA GGC TTA GAA GGT AAA GTA AAA TGG-3′ for the 10Ala and 17Ala constructs of generic construct B, 5′-AGC CAT ATG GCA GGA GGA GGC TTA GAA GGT AAA GTA AAA TGG-3′ for the deletion (ΔAla) variant of construct B and 5′-GAA GAC ATT TAG TAC CAG GAT TAG AAG GTA AAG TAA AAT GG-3′ for the generic construct A (16 residue linker). The reverse primers had the reverse complementary sequences. PCR products were treated with DpnI and transformed in E. coli XL1-Blue. Similarly, the A46K/S48R mutation was introduced into the coding DNA for CspB via the QuickChange II Site-Directed Mutagenesis Kit. For this mutagenesis, the primer 5′-CTT TAG AAG AAG GCC AAA AAG TTC GTT TTG AAA TCG TTG AAG G-3′ and its reverse complement were used. Recombinant gene expression and protein purification was performed according to For production of variants with linkers of 3 or 16 amino acids, a fusion construct consisting of the N-terminal domain of PABPN1 and CspB served as a template −1 cm−1 for generic constructs A, B and C Protein purity and concentrations of the soluble species were assessed by UV spectroscopy recording readings at 280 nm. The molar extinction coefficients were determined as 5500 Mg in an Optima TM TLX ultracentrifuge, washed with 2 M (10Ala-CspB) or 1.5 M (17Ala-L3/L16-CspB) guanidinium chloride (GdmCl), 5 mM KH2PO4, 100 mM NaCl, pH 7.5 and again separated by centrifugation. Fibrils were resuspended in 50 mM Tris/HCl, 100 mM NaCl, 2 mM CaCl2, pH 8.0, and the protein concentration was adjusted to 1 to 5 mg/ml. Proteinase K was added at a ratio of 1∶50 (w/w), and the solution was incubated for 2 h at 37°C. Reactions were stopped by adding PMSF. After centrifugation for 1 h at 260 000 g, the pellet was first washed with 2 M or 1.5 M GdmCl, 5 mM KH2PO4, 100 mM NaCl, pH 7.5, respectively and then twice with 5 mM KH2PO4, 100 mM NaCl, pH 7.5. Fibrils of 10Ala-CspB were solubilized with 5 M guanidinium thiocyanate (GdmSCN), 0.1% trifluoroacetic acid (TFA) (v/v) over night at room temperature. The solution was again centrifuged, and the supernatant analyzed by reversed phase HPLC. Peptides were separated on a Nucleosil 5u C18 (125×4 mm 5 micron) column  at a flow rate of 0.7 ml min−1. After injection of the solution, the column was flushed with H2O, 0.05% TFA (v/v) for 3 min. Peptides were eluted with an increasing gradient of acetonitril (AcN), 0.05% TFA (v/v) (0–50% in 70 min and 50–100% in 17 min). The separated peptides were analyzed by MALDI-TOF mass spectrometry using a Reflex II-mass spectrometer . For the alignment of the proteolytic fragments, the program FindPept (http://www.expasy.ch/tools/findpept.html) was used. Fibrils of 17Ala-L3/L16-CspB were solubilized with 6 M GdmCl, 5 mM KH2PO4, 100 mM NaCl, pH 7.5 and analyzed by fluorescence spectroscopy in the same buffer.Proteolysis of the fibrils was performed with proteinase K. Fibrils were harvested by centrifugation for 1 h at 260 000 2PO4, 100 mM NaCl, pH 7.5 at 20°C at protein concentrations of 0.85 to 1.1 mg/ml in 0.1 mm cuvettes. The spectra were measured 10 times and averaged. Data were collected in 0.1 nm steps. Spectra were buffer corrected. Tryptophan fluorescence was recorded with a Jobin Yvon Fluoromax2 equipped with a thermostatted cell holder. Measurements were performed in 5 mM KH2PO4, 100 mM NaCl, pH 7.5 at 20°C at a protein concentration of 3 µM. The excitation wavelength was 295 nm at a bandwidth of 5 nm. Spectra were recorded in 1 cm cuvettes at a bandwidth of 5 nm and accumulated three times. Data were averaged and buffer corrected.CD spectra were recorded with a Jasco J810 spectropolarimeter in 5 mM KH2PO4, 100 mM NaCl, pH 7.5, at protein concentrations of 0.15 to 0.22 mg/ml in 0.1 cm cuvettes. The transitions were monitored by changes of the CD signals at 205 nm at 1 nm band width. Heating rates were 1°C min−1. Transitions were evaluated using a non-linear least-square fit according to a two state model −1 K−1 was used for the change in heat capacity upon unfolding (ΔCP). For urea-induced unfolding, urea  concentrations were determined by refraction 2PO4, 100 mM NaCl, pH 7.5 containing various urea concentrations. Denaturation was allowed to proceed for 1 h at 20°C. Fluorescence was recorded at an emission wavelength of 345 nm upon excitation at 295 nm with a Jobin Yvon Fluoromax2 at 20°C. Experiments were performed in 1 cm cuvettes with excitation and emission band width of 5 nm each. The unfolding transitions were analyzed by assuming a two-state transition between the folded (N) and unfolded (U) conformation. For obtaining the thermodynamic parameters ΔGD and m values, non-linear least-square fits were performed Thermal unfolding transitions were measured in 5 mM KH5′-TTTTTTT-3′ was purchased from Thermo Electron GmbH (Germany). Concentrations of dT7 were calculated by the absorbance at 260 nm. The extinction coefficient of dT7 at 260 nm was determined as 58 800 M−1 cm−1 according to The single-stranded DNA fragment dT7 2PO4, 100 mM NaCl, pH 7.5 containing 1 µg/ml NaN3. Seeded reactions contained 5% (w/w) seeds prepared from fibrils of 10Ala-CspB. Seeds were prepared as described previously 2PO4, 100 mM NaCl, pH 7.5 at final concentrations of 5 µM. Fluorescence was recorded with a Jobin Yvon Fluoromax2 upon excitation at 370 nm and at an emission wavelength of 480 nm at 20°C. Experiments were performed in 1 cm cuvettes with an excitation and emission slit width of 5 nm. For EM analysis, carbonized copper grids  were used, prepared as published Proteins were dissolved to final concentrations of 0.5 mM and incubated at 37°C. The buffer was 5 mM KHIn previous work we had shown that when CspB is fused with the complete N-terminal domain of PABPN1 fibrils are formed in which the CspB moiety remained folded. In contrast, in fibrils arising from “direct fusions” between the oligo-Ala stretch and CspB  and the length of the linker (3 or 16 residues) were varied. In addition, control proteins were produced, in which only the 3- or 16-residues linkers, but not the oligo-alanine stretches were fused to CspB. In the nomenclature of the variants, “10Ala” and “17Ala” denote the length of the amyloidogenic alanine stretch, “L3” and “L16” denote the length of the linker to the CspB moiety . These lKD values for all variants. They show that the high affinity of CspB for the oligonucleotide dT7 is retained in all fusion proteins. Thus, CspB was correctly folded and functional  of 12.4 kJ/mol under the conditions of our stability measurements GD was reduced to 6.0 kJ/mol  or ThT (Thioflavin T) fluorescence and electron microscopy (EM). As shown earlier by us and others for oligo-Ala fibrils, ANS fluorescence changes proved to be more sensitive for monitoring changes upon fibrillization than ThT fluorescence measurements , and, at 25°C, about one out of 150 CspB molecules is in the unfolded state. The stability is decreased further when the CspB moiety is fused with amyloidogenic segments. This, together with the high rate of unfolding of CspB The conformational stability of isolated wild-type CspB is low Click here for additional data file.Figure S1Coomassie-stained SDS-polyacrylamide gel of the purified fusion variants.(TIF)Click here for additional data file.Figure S2Urea-induced refolding transitions of the fusions with CspB and MCspB. Variants with 10 alanines are shown with filled; variants with 17 alanines with open symbols; variants with linkers of 3 amino acids are shown as circles; variants with linkers of 16 amino acids as triangles. Measurements were carried out in 5 mM KH2PO4, 100 mM NaCl, pH 7.5 at 20°C. A Transitions of the fusions with CspB, B Transitions of the fusions with MCspB.(TIF)Click here for additional data file.Figure S3Comparison of ANS and ThT fluorescence signals of 17Ala-L16-CspB during fibril formation. Filled symbols correspond to ThT, open symbols to ANS signals.(TIF)Click here for additional data file.Figure S4ANS fluorescence of fusion proteins without alanines. The fusions were incubated at concentrations of 0.5 mM in 5 mM KH2PO4, 100 mM NaCl, pH 7.5 at 37°C. ΔAla-L16-CspB, filled triangles; ΔAla-L3-CspB, filled circles.(TIF)Click here for additional data file.Figure S5Characterization of the variants with MCspB by far-UV-CD (A) and dT7 binding (B). In A, variants with 10 alanines are black, variants with 17 alanines are gray; variants with the L3 linker, dashed line and with L16 linker, dotted-dashed line. As a reference, the spectrum of MCspB is shown in the black, solid line. In B, variants with 10 alanines are shown by filled symbols, variants with 17 alanines by open symbols; variants with L3 linkers are indicated by circles, variants with L16 linkers by triangles. The corresponding KD values are listed in 2PO4, 100 mM NaCl, pH 7.5 at 20°C.(TIF)Click here for additional data file.


Escherichia coli usually offer between 95 to 99% of protein recovery, what in practical terms, might imply extensive bacterial cell contamination, not compatible with the use of inclusion bodies in biological interfaces.Bacterial inclusion bodies are submicron protein clusters usually found in recombinant bacteria that have been traditionally considered as undesirable products from protein production processes. However, being fully biocompatible, they have been recently characterized as nanoparticulate inert materials useful as scaffolds for tissue engineering, with potentially wider applicability in biomedicine and material sciences. Current protocols for inclusion body isolation from -1 cfu/ml, keeping the particulate organization of these aggregates regarding size and protein folding features.Using an appropriate combination of chemical and mechanical cell disruption methods we have established a convenient procedure for the recovery of bacterial inclusion bodies with undetectable levels of viable cell contamination, below 10The application of the developed protocol allows obtaining bacterial free inclusion bodies suitable for use in mammalian cell cultures and other biological interfaces. Bacterial inclusion bodies (IBs) are water-insoluble protein aggregates formed in the bacterial cytoplasm  during the overproduction of recombinant proteins, especially those from viral or mammalian origin . The diaOn the other hand, a wide range of protocols for the purification of cytoplasmic IBs are available, all of them including bacterial lysis and IB washing steps. Bacterial lysis is achieved using either mechanical or non mechanical methods, or a combination of both, while washing steps include, among others, detergent and/or DNase treatments. These protocols have been mainly aimed to obtain IBs suitable for in vitro protein refolding attempts . Since mThe aim of this study has been the development of an IB purification protocol rendering bacterial-free protein particles, which could not compromise the applicability of IBs as biomaterials for biomedical applications.- and ClpP-) carrying plasmids encoding different recombinant proteins   Figure . Compari00, DnaK Escherichia coli strains overproducing VP1GFP needed 5 sonication rounds of 10 min at 40% amplitude under 0.5 s cycles, independently of the genetic background  U169 rpsL150 relA1 flbB5301 deoC1 ptsF25 rbsR, StrepR) [clpP::cat StrepR) and JGT20  [The  StrepR)  and theipR, TcR) . The strpR, TcR) . JGT10 aBacterial strains were cultured in shake flask cultures at 37°C and 250 rpm in LB rich medium  plus 100The concentration of colony forming units (cfu/ml) was determined on LB plates with the corresponding antibiotics. After an appropriate dilution in Ringer 1/4, samples were inoculated on LB plates and incubated at 37°C o/n. Cell counting was always performed in triplicate.Culture samples of 20 ml were taken 3 h after induction and IBs were purified by using two different purification protocols as follows.Lysozyme and repeated detergent washing treatment: cells were harvested by centrifugation at 15,000 g at 4°C for 15 min and resuspended in 400 μl of lysis buffer (50 mM TrisHCl (pH 8.1), 100 mM NaCl and 1 mM EDTA,) and kept at -80°C o/n. After thawing, phenylmethanesulphonylfluoride (PMSF)  and lysozyme  were added. After 45 min of incubation at 37°C, 4 μl of Nonidet P40 (NP-40) were added and the mixture incubated at 4°C for 1 h. Then, 12 μl of DNase I (from a 1 mg/ml stock) and 12 μl of 1 M MgSO4 were added and the resulting mixture was further incubated at 37°C for 45 min. Protein aggregates were separated by centrifugation at 15,000 g for 15 min at 4°C. Finally, IBs were washed once with 1 ml of the same lysis buffer containing 0.5% Triton X-100. After a final centrifugation at 15,000 g for 15 min at 4°C, pellets were stored at -80°C until analysis. All incubations were done under gentle agitation.Lysozyme-detergent, sonication and repeated detergent washing treatment: samples of bacterial cultures (20 ml) were centrifuged at 4°C at 5,000 g for 5 min and resuspended in lysis buffer , 100 mM NaCl, and 1 mM EDTA) and frozen at -80°C o/n. After thawing, phenylmethanesulphonylfluoride (PMSF)  and 1 mg/ml lysozyme  were added. After 2 h of incubation at 37°C, 100 μl of Triton X-100 were added (0.5% Triton X-100) and incubated at room temperature for 1 h. Then, the mixture was ice-jacketed, and sonicated between 4 and 10 cycles of 10 min at 40% amplitude under 0.5 s cycles. After sonication, an aliquot of 100 μl of the suspensions were inoculated on LB plates with the corresponding antibiotics and incubated at 37°C o/n. After that, 5 μl of Nonidet P40 (NP-40) were added to the rest of the suspension, and samples incubated at 4°C for 1 h. Then, DNA was removed with DNase  and MgSO4  for 45 min at 37°C. Finally, samples were centrifuged at 4°C at 15,000 g for 15 min, and the pellet containing pure IBs was washed once with 1 ml of lysis buffer containing Triton X-100 (0.5%). After a final centrifugation at 15,000 g for 15 min at 4°C, pellets were stored at -80°C until analysis. All incubations were done under agitation.At 3 h post-induction, VP1GFP-producing cells were fixed with 0.1% formaldehyde in phosphate buffered saline (PBS) and purified IBs were also resuspended in PBS and stored at 4°C until observed. Samples of bacterial cells or IBs were placed on a glass slide, fixed with a slide cover and observed with a Leica TCS SP2 AOBS confocal fluorescence microscope  using a Plan-Apochromat objective  and optical lens magnification . Photomicrographs were obtained after excitation at 488 nm and at emission wavelengths between 500 and 600 nm.2HPO4 0.9 M, Na2H2PO4 0.06 M, pH 8.0) for 1 h at 4°C. After that, the samples were dehydrated with increasing concentrations of ethanol in water  by consecutive 5 min washing steps. Ethanol was finally evaporated using the critical point method in a K850 CPD desiccator . The dried membranes were sputtered with gold using a K550 Sputter Coater  for observation. Microscopy was performed with a scanning microscope Hitachi S-570  using an acceleration between 0.5-30 kV.Bacterial samples and purified IBs were retained on a nuclepore membrane  and fixed with 2.5% phosphate buffered glutaraldehyde (NaThe authors declare that they have no competing interests.ERC performed most of the experiments and prepared the final data and figures. OCG and JSF purified inclusion bodies and analysed samples by fluorescence microscopy. AV and EGF conceived of the study. EGF directed the work and prepared the manuscript. All authors read and approved the final manuscript.


Mycobacterium tuberculosis. In addition, we examined if the antigen dose could influence the phenotype of the induced T cells.Previously we have shown that Ag85B-TB10.4 is a highly efficient vaccine against tuberculosis when delivered in a Th1 inducing adjuvant based on cationic liposomes. Another Th1 inducing adjuvant, which has shown a very promising profile in both preclinical and clinical trials, is IC31®. In this study, we examined the potential of Ag85B-TB10.4 delivered in the adjuvant IC31® for the ability to induce protection against infection with M.tb in the mouse TB model. Importantly, our results also showed that both the vaccine induced T cell response, and the protective efficacy, was highly dependent on the antigen dose. Thus, whereas antigen doses of 5 and 15 µg did not induce significant protection against M.tb, reducing the dose to 0.5 µg selectively increased the number of polyfunctional T cells and induced a strong protection against infection with M.tb. The influence of antigen dose was also observed in the guinea pig model of aerosol infection with M.tb. In this model a 2.5 fold increase in the antigen dose reduced the protection against infection with M.tb to the level observed in non-vaccinated animals.We found that vaccination with the combination of Ag85B-TB10.4 and IC31® resulted in high numbers of polyfunctional CD4 T cells co-expressing IL-2, IFN-γ and TNF-α. This correlated with protection against subsequent challenge with Mycobacterium tuberculosis. This vaccine has now entered clinical trials.Small changes in the antigen dose can greatly influence the induction of specific T cell subpopulations and the dose is therefore a crucial factor when testing new vaccines. However, the adjuvant IC31® can, with the optimal dose of Ag85B-TB10.4, induce strong protection against  M.tbM.tb5KLK and the immunostimulatory oligodeoxynucleotide ODN1a that signals through the TLR9 receptor. The combination of Ag85B-TB10.4 and IC31® is attractive for several reasons; 1) IC31® has been shown to promote a strong Th1 response and was demonstrated to have a very compelling safety profile in the first clinical trial, and 2) Ag85B-TB10.4 has the advantage that it does not include ESAT-6, which is a valuable diagnostic reagent and the basis of a number of commercial diagnostic tests M.tb. Our results show that Ag85B-TB10.4/IC31® can induce high numbers of polyfunctional CD4 T cells and induce efficient protection against infection with M.tb. However, the combination of the immunogenic Ag85B-TB10.4 antigen and the IC31® adjuvant activity was very sensitive to the dose of antigen. Thus, whereas the higher doses antigen of Ag85B-TB10.4 (5 and 15 µg) in IC31®, induced very modest immune responses that did not protect against M.tb challenge, reducing the dose to 0.5 µg Ag85B-TB10.4 in IC31® led to a selective increase in the numbers of polyfunctional T cells, which in turn increased the protection against M.tb to the same level as BCG. Also in the Guinea pig TB animal model we observed a strong correlation between vaccine efficacy and antigen dose. Thus, we found that decreasing or increasing the antigen dose as little as 2.5 fold compared to the optimal dose led to a significantly reduced protective efficacy of the vaccine.The global effort to develop an effective TB vaccine involves different strategies including live attenuated vaccines in vitro stimulation of purified PBMCs with Ag85B or TB10.4 (or ESAT-6 as a negative control) . The antigen dose used in the experiment was in the range from 0.005 to 15 µg. One week after the last injection, the mice were bled, and the IFN-γ release was evaluated after control) . Vaccinacontrol) . The samcontrol)  where thcontrol) .+IFN-γ+IL-2+TNF-α+ and CD4+IL-2+TNF-α+ T cells. Similarly to in the result depicted in We next analyzed the cytokine expression of the T cells induced by immunizing with 0.5 µg compared to 5 µg Ag85B-TB10.4/IC31®. In particular, we were interested in analyzing the induction of polyfunctional CD4 T cells as these have been shown to correlate with protective immunity against infections such as Leishmania major and to form the basis for a long lived memory response M. tuberculosis and bacterial numbers were assessed in the lungs six weeks post challenge. As observed with the immunogenicity of the vaccine, the protective efficacy of the vaccine also decreased when vaccination dose was increased from 0.5 µg to 5 or 15 µg , but significantly reduced (p<0.001) compared to the bacterial numbers in non-vaccinated mice (5.83+/−0.12 Log10 CFU)  . This wag10 CFU) . Repeatig10 CFU) .M.tb correlated with the quantity and quality of the T cell response induced, and in particular with the proportion of polyfunctional T cells that co-express all three cytokines, which was highest when the lowest antigen dose was used.In conclusion, the ability of the vaccine, Ag85B-TB10.4/IC31®, to induce protection against M. tuberculosis, and survival was monitored (based on weight loss). The results showed that there was no significant difference in survival between adjuvant treated guinea pigs and those receiving 0.1 or 50 µg of vaccine  using an antigen dose of 0.1 ,1.0, 10, 20 and 50 µg. 10 weeks after the last vaccination the animals were infected via the aerosol route with virulent  vaccine . HoweverM.tb. Surprisingly, compared to a standard dose of 5 µg antigen, reducing the dose down to 0.005 µg (×1000 fold reduction) led to a stronger immune response directed to both vaccine components  mice from Harland Netherlands. Infected animals were housed in cages contained within laminar flow safety enclosures in a BSL-3 facility. All mice were fed radiation sterilized 2016 Global Rodent Maintenance diet  and water ad libitum. All animals were allowed a one-week rest before initiation of the experiments. Out-bred Hartley guinea pigs  were infected and housed in cages contained within a BL-3 laminar flow safety enclosure. Guinea pigs were weighed weekly and were euthanized based on weight loss, increased respiratory rate (laboured/heavy breathing) and general behaviour and appearance.Mycobacterium tuberculosis Erdman were grown at 37°C on Löwenstein-Jensen medium or in suspension in Sauton medium enriched with 0.5% sodium pyruvate and 0.5% glucose. M. tuberculosis H37Rv (TMC#102) for guinea pig infection studies was grown in Proskaur & Beck medium as described previously 5 CFU) injected subcutaneously at the base of the tail and one group received a saline injection. All groups of mice were challenged 10 weeks after the first vaccination.Mice were vaccinated three times at 2-week intervals subcutaneously on the back with experimental vaccines containing 0.005, 0.05, 0.5, 5 or 15 µg of Ag85B-TB10.4 (H4)/dose, emulsified in IC31® in a total volume of 0.2 ml/dose. The adjuvant IC31®consists of a mixture of the peptide KLK (NH2-KLKL5KLK-COOH) and the oligodeoxynucleotide ODN1a (oligo-(dIdC)13) provided by Intercell. Doses were 100 nmol peptide and 4 nmol oligonucleotide. All vaccines were formulated using 10 mM Tris-HCL/270 mM sorbitol buffer (pH 7.9) as previously described M. tuberculosis Erdman/mouse. These mice were sacrificed 6 weeks after challenge. Numbers of bacteria in the spleen or lung were determined by serial threefold dilutions of individual whole-organ homogenates in duplicate on 7H11 medium. Organs from the BCG-vaccinated animals were grown on medium supplemented with 2 µg of 2-thiophene-carboxylic acid hydrazide (TCH)/ml to selectively inhibit the growth of the residual BCG bacteria in the test organs. Colonies were counted after 2 to 3 weeks of incubation at 37°C. Bacterial burden in the lungs was expressed as log10 coloni forming units (CFU).When challenged by the aerosol route, the mice were infected with approximately 50 CFU of 5 cells in a volume of 200 µl of RPMI 1640 supplemented with 5×10−5 M 2-mercaptoethanol, 1% penicillin-streptomycin, 1 mM glutamine, and 5% (vol/vol) fetal calf serum. Based on previous dose-response investigations, the mycobacterial antigens were all used at 1 µg/ml. All preparations were tested in cell cultures and found to be nontoxic at the concentrations used in the present study. Supernatants were harvested from cultures after 72h of incubation for the investigation of IFN-γ.Lymphocytes from spleens were obtained as described previously Microtiter plates  were coated with monoclonal hamster anti-murine IFN-γ  in PBS at 4°C. Free binding sites were blocked with 1% (wt/vol) bovine serum albumin-0.05% Tween 20. Culture supernatants were tested in triplicate, and IFN-γ was detected with a biotin-labelled rat anti-murine monoclonal antibody . Recombinant IFN-γ  was used as a standard.Intracellular cytokine staining procedure: Cells from blood, spleen or lungs of mice were stimulated for 1–2 h with 2 µg/ml Ag at 37°C and subsequently incubated for 5 h at 37°C with 10 µg/ml brefeldin A  at 37°C. Fc receptors were blocked with 0.5 ìg/ml anti-CD16/CD32 mAb  for 10 minutes, whereafter the cells were washed in FACS buffer (PBS containing 0.1% sodium azide and 1% FCS) before staining with a combination of the following rat anti-mouse antibodies PE-Cy7-, PerCP-Cy5.5-anti-CD8á , APC-Cy7-anti-CD4 (GKI.5) . Cells were washed with FACS buffer before fixation and permeabilization using the BD Cytofix/Cytoperm™  according to the manufacturer's protocol before intracellular staining with PE-, PE-Cy7, APC-Anti-IFN-ã (XMG1.2), PE-anti-TNFα, and/or PE-, APC-anti-IL-2 (JES6-5H4). After washing, cells were resuspended in formaldehyde solution 4% (w/v) pH 7.0  and samples were analyzed on a six-color BD FACSCanto flow cytometer . Data analysis was done with FACSDiva Software  and Flowjo Software .6 spleen cells.96-well microtiter plates  were coated with 4 µg/ml monoclonal rat anti-murine IFN-γ . Free binding sites were blocked with RMPI 1640 supplemented as described above for lymphocyte cul-tures. After 48 hrs of incubation with 2 µg/ml Ag at 37°C IFN-γ was detected with 1.25 µ/ml biotin labeled rat anti-murine Ab  and 0.5 µg/ml al-kaline phosphatase-conjugated streptavidin (Jackson ImmunoResearch Laboratories Europe). The enzyme reaction was developed with SIGMA FAST™ BCIP/NBT (5-Bromo-4-chloro-3-indolyl phosphate/Nitro blue tetrazolium)  and stopped by washing the plates with ddH2O. ELISPOT plates were counted using an AID plate reader  and AID ELISPOT 3.0c software. IFN-γ-specific cells expressed as number of spot-forming units (SFU)/10The data obtained were tested by analysis of variance. Differences between means were assessed for statistical significance by Tukey's test. A P value of <0.05 was considered significant. The Log Rank tested was used to analyze guinea pig survival data.