Calcium Signaling Controls Pathogenic Th17 Cell-Mediated Inflammation by Regulating Mitochondrial Function

1. Download : Download high-res image (229KB)
2. Download : Download full-size image

     

Somatic Mutations in Exocrine Pancreatic Tumors: Association with Patient Survival

KRAS mutations are major factors involved in initiation and maintenance of pancreatic tumors. The impact of different mutations on patient survival has not been clearly defined. We screened tumors from 171 pancreatic cancer patients for mutations in KRAS and CDKN2A genes. Mutations in KRAS were detected in 134 tumors, with 131 in codon 12 and only 3 in codon 61. The GGT>GAT (G12D) was the most frequent mutation and was present in 60% (80/134). Deletions and mutations in CDKN2A were detected in 43 tumors. Analysis showed that KRAS mutations were associated with reduced patient survival in both malignant exocrine and ductal adenocarcinomas (PDAC). Patients with PDACs that had KRAS mutations showed a median survival of 17 months compared to 30 months for those without mutations (log-rank P = 0.07) with a multivariate hazard ratio (HR) of 2.19 (95%CI 1.09–4.42). The patients with G12D mutation showed a median survival of 16 months (log-rank-test P = 0.03) and an associated multivariate HR 2.42 (95%CI 1.14–2.67). Although, the association of survival in PDAC patients with CDKN2A aberrations in tumors was not statistically significant, the sub-group of patients with concomitant KRAS mutations and CDKN2A alterations in tumors were associated with a median survival of 13.5 months compared to 22 months without mutation (log-rank-test P = 0.02) and a corresponding HR of 3.07 (95%CI 1.33–7.10). Our results are indicative of an association between mutational status and survival in PDAC patients, which if confirmed in subsequent studies can have potential clinical application.     

A “new” genetic polymorphism of a human serum protein: inter-alpha-trypsin-inhibitor

Summary A new genetic polymorphism of a human serum glycoprotein, the inter--trypsin-inhibitor (ITI), has been demonstrated by population and family studies. Sera were examined after neuraminidase treatment by isoelectric focusing on agarose gels followed by immunoblotting or by immunfixation with specific ITI-antiserum. Using this method, three common ITI phenotypes 1, 1–2 and 2, as well as two further rare ITI types 1–3 and 2–3 were disclosed. Genetically, these phenotypes are controlled by three allelic genes that determine a total of six phenotypes. These alleles are designated ITI^*1, ITI^*2 and ITI^*3. The homozygous form of the third allele ITI^*3 has not been found, as yet. The frequencies of ITI were examined in two population samples from Southern Germany (n=248) and from Tyrol, Austria (n=124). The gene frequencies of the common alleles ITI^*1 and ITI^*2 were 0.575 and 0.417, respectively, in Southern Germany, and 0.577 and 0.423, respectively, in Tyrol, Austria. The third allele ITI^*3 was found only in the sample from Southern Germany, thus far, and was calculated to be 0.008.     

Female mice carrying a defective Alox15 gene are protected from experimental colitis via sustained maintenance of the intestinal epithelial barrier function

Lipoxygenases (ALOXs) are involved in the regulation of cellular redox homeostasis. They also have been implicated in the biosynthesis of pro- and anti-inflammatory lipid mediators and play a role in the pathogenesis of inflammatory diseases, which constitute a major health challenge owing to increasing incidence and prevalence in all industrialized countries around the world. To explore the pathophysiological role of Alox15 (leukocyte-type 12-LOX) in mouse experimental colitis we tested the impact of systemic inactivation of the Alox15 gene on the extent of dextrane sulfate sodium (DSS) colitis. We found that in wildtype mice expression of the Alox15 gene was augmented during DSS-colitis while expression of other Alox genes (Alox5, Alox15b) was hardly altered. Systemic Alox15 (leukocyte-type 12-LOX) deficiency induced less severe colitis symptoms and suppressed in vivo formation of 12-hydroxyeicosatetraenoic acid (12-HETE), the major Alox15 (leukocyte-type 12-LOX) product in mice. These alterations were paralleled by reduced expression of pro-inflammatory gene products, by sustained expression of the zonula occludens protein 1 (ZO-1) and by a less impaired intestinal epithelial barrier function. These results are consistent with in vitro incubations of colon epithelial cells, in which addition of 12S-HETE compromised enantioselectively transepithelial electric resistance. Consistent with these data transgenic overexpression of human ALOX15 intensified the inflammatory symptoms. In summary, our results indicate that systemic Alox15 (leukocyte-type 12-LOX) deficiency protects mice from DSS-colitis. Since exogenous 12-HETE compromises the expression of the tight junction protein ZO-1 the protective effect has been related to a less pronounced impairment of the intestinal epithelial barrier function.     

Auditory memory for temporal characteristics of sound

This study evaluates auditory memory for variations in the rate of sinusoidal amplitude modulation (SAM) of noise bursts in the European starling (Sturnus vulgaris). To estimate the extent of the starling’s auditory short-term memory store, a delayed non-matching-to-sample paradigm was applied. The birds were trained to discriminate between a series of identical “sample stimuli” and a single “test stimulus”. The birds classified SAM rates of sample and test stimuli as being either the same or different. Memory performance of the birds was measured as the percentage of correct classifications. Auditory memory persistence time was estimated as a function of the delay between sample and test stimuli. Memory performance was significantly affected by the delay between sample and test and by the number of sample stimuli presented before the test stimulus, but was not affected by the difference in SAM rate between sample and test stimuli. The individuals’ auditory memory persistence times varied between 2 and 13 s. The starlings’ auditory memory persistence in the present study for signals varying in the temporal domain was significantly shorter compared to that of a previous study (Zokoll et al. in J Acoust Soc Am 121:2842, 2007) applying tonal stimuli varying in the spectral domain.     

Circular dichroism and guanine quadruplexes

Circular dichroism (CD) is remarkably sensitive to the conformational states of nucleic acids; therefore, CD spectroscopy has been used to study most features of DNA and RNA structures. Quadruplexes are among the significant noncanonical nucleic acids architectures that have received special attentions recently. This article presents examples on the contribution of CD spectroscopy to our knowledge of quadruplex structures and their polymorphism. The examples were selected to demonstrate the potential of this simple method in the quadruplex field. As CD spectroscopy detects only the global feature of a macromolecule, it should preferably be used in combination with other techniques. On the other hand, CD spectroscopy, often as a pioneering approach, can reveal the formation of particular structural arrangements, to search for the conditions stabilizing the structures, to follow the transitions between various structural states, to explore kinetics of their appearance, to determine thermodynamic parameters and also detect formation of higher order structures. This article aims to show that CD spectroscopy is an important complementary technique to NMR spectroscopy and X-ray diffraction in quadruplex studies.     

Gag p27-Specific B- and T-Cell Responses in Simian Immunodeficiency Virus SIVagm-Infected African Green Monkeys

Nonpathogenic simian immunodeficiency virus SIVagm infection of African green monkeys (AGMs) is characterized by the absence of a robust antibody response against Gag p27. To determine if this is accompanied by a selective loss of T-cell responses to Gag p27, we studied CD4⁺ and CD8⁺ T-cell responses against Gag p27 and other SIVagm antigens in the peripheral blood and lymph nodes of acutely and chronically infected AGMs. Our data show that AGMs can mount a T-cell response against Gag p27, indicating that the absence of anti-p27 antibodies is not due to the absence of Gag p27-specific T cells.Simian immunodeficiency virus (SIV) infection in African green monkeys (AGM) is nonpathogenic, even though it is characterized by plasma viral load (PVL) levels similar to those found during acute and chronic pathogenic infection of humans with human immunodeficiency virus type 1 and macaques with SIVmac (14). This feature is shared with other African nonhuman primates, such as sooty mangabeys (SM) and mandrills (19, 20). SIV-infected AGMs also display high viral loads in the gastrointestinal mucosa (11), a transient decline of circulating CD4⁺ T cells during acute infection (13), and longer-lasting CD4⁺ T-cell depletion in the intestinal lamina propia (10). Concomitant with the peak viral load during acute infection, SIVagm-infected AGMs display transient increases of CD4⁺ and CD8⁺ T cells expressing activation, and proliferation markers, such as MHC-II DR and Ki-67 (4, 13), and anti-SIVagm antibodies (Ab) are induced with kinetics similar to those found in SIVmac infection (5). Interestingly, however, the Ab response against Gag p27 is weak, if present at all (1, 2, 12, 15, 17, 18). This observation is surprising since, in the context of human immunodeficiency virus type 1 and SIVmac infections, Ab responses to Gag p27 are usually quite strong. Weak or low reactivity to Gag p27 has also been observed in some other natural SIV infections (7, 8, 20) but not in all of them (21). We wondered whether such a selective lack of Ab reactivity in the SIV-infected AGM might be related to a lack of Gag p27-specific T cells. With this hypothesis in mind, we first confirmed and extended the studies of humoral responses against Gag p27 by characterizing the antigen-specific immunoglobulin G (IgG) responses and mid-point titers against total SIVagm antigens (SIVagm virions) and recombinant Gag p27 (rP27; SIVagm) in naturally and experimentally SIVagm-infected AGMs. Second, we searched for the presence of Gag p27-specific T-cell responses in SIVagm infection by analyzing the CD4⁺ and CD8⁺ T-cell responses specific for Gag p27 and other SIVagm proteins in blood and lymph nodes (LNs) of acutely and chronically infected animals.Humoral responses against SIV were analyzed in 50 wild-born AGMs (Chlorocebus sabaeus) and 17 rhesus macaques (RMs). The animals were housed at the Institut Pasteur in Dakar, Senegal, and the California National Primate Research Center, Davis, CA, respectively, according to institutional and national guidelines. RMs were either noninfected (n = 5) or intravenously infected with SIVmac251 (n = 12). AGMs were noninfected (n = 23), naturally infected (n = 17), or intravenously infected with wild-type SIVagm.sab92018 (n = 10) (5, 9). IgG titers against SIVagm.sab92018 virions or rP27 were determined by an enzyme-linked immunosorbent assay (ELISA) using monkey anti-IgG as secondary Ab (Fig. 1A and B). The virions had been purified by ultracentrifugation on an iodixanol cushion from cell-free supernatants of SIVagm.sab92018-infected SupT1 cells. The His-tagged rP27 was constructed using DNA from gut cells of an SIVagm.sab92018-infected AGM 96011 (11). A Gag p27 PCR product was subcloned into pET-14b, and the recombinant protein was produced in Escherichia coli BL21(DE3)(pLysS) and purified on nitrilotriacetic acid columns. SIV-infected macaques showed high IgG titers cross-reacting with both SIVagm virions (Fig. 1A and B, left panels) and rP27 (Fig. 1A and B, right panels). In contrast, only 2 out of 27 SIV-infected AGMs showed detectable IgG responses against rP27 (Fig. 1A and B, right panels), while 21 out of 27 displayed significant responses against SIVagm virions (Fig. 1A and B, left panels). Two AGMs out of 23 from the negative control group showed weak responses at the limit of detection against SIVagm and two against rP27, suggesting a natural response against SIVagm proteins, cross-reactivity with unknown pathogens, maternal Ab, or recent SIV infection. Of note, the titers against whole SIV in the infected monkeys were higher in macaques than in AGMs, which may be due to a lack of anti-p27 Ab in most AGMs.Open in a separate windowFIG. 1.Cross-sectional analysis of IgG Ab responses against SIVagm or Gag p27 in SIV-infected AGMs and RMs. (A and B) Cross-sectional analysis by ELISA. IgG Ab against SIVagm.sab₉₂₀₁₈ virions or recombinant p27-Gag antigens were determined in SIV-negative (Rh SIV−) and chronically SIVmac₂₅₁-infected (Rh SIV+) RMs and in SIV-negative and chronically SIVagm-infected AGMs that were either naturally (AGM Nat SIV+) or experimentally (AGM Exp SIV+) infected with SIVagm.sab₉₂₀₁₈. Ab titers were calculated for each animal by limited dilution of plasma on coated ELISA plates with 5 μg/ml of (p27 equivalent) virions (left) or 1 μg/ml of the monomeric recombinant protein (rP27) (right). IgG detection by ELISA displayed a high background for rP27, especially at the highest plasma concentration (e.g., 1/100 and 1/400 plasma dilution) in SIV-negative RMs and AGMs. To discriminate between positive responses and background, calculated dose-response curves were compared to theoretical sigmoid-dose response curves corresponding to the 95% confidence interval of SIV-negative animals. By convention, responses were considered background when sigmoid dose-response curves were graphically within the 95% confidence interval of SIV-negative animals and when the calculated negative log 50% effective concentration (EC₅₀) was lower than the top theoretical sigmoid dose-response curve from SIV-negative animals (corresponding to a threshold of negative log EC₅₀ of 2.8). (A) Results (optical density at 450 nm [OD₄₅₀]) are represented for both virions (left) and rP27 (right) over plasma dilution (log₁₀) on a per animal basis (data points) and for each group (lines). Lines represent the sigmoid dose-response curves for each group (Prism 4; Graphpad). (B) Mid-point IgG titers were determined for each animal from individual sigmoid dose-response curves, and presented as the log₁₀ value from the reciprocal of the effective concentration that corresponds to 50% response between minimum and maximum OD₄₅₀ (negative log EC₅₀). Horizontal bars represent the median mid-point titer per each group. Mann-Whitney nonparametric tests were applied for statistical analysis (n.s., nonsignificant, with P values of >0.1) (C) Cross-sectional analysis of Ab against SIVagm proteins by Western blot analysis using denatured SIVagm.sab₉₂₀₁₈. For the positive controls on the left, we used sera from an SIVmac₂₅₁-infected macaque and a SIVagm.sab₉₂₀₁₈-infected AGM. Development times and reagents were identical for all Western blots. Mo, months of infection; y, years of infection; C−, negative control; C+, positive control.The study of IgGs by Western blot analysis using denatured SIVagm.sab92018 virions showed no or weak anti-Gag responses in SIV-infected AGMs, yet the anti-Env responses were often strong (Fig. ​(Fig.1C).1C). In contrast, SIV-infected macaques showed a dominant IgG cross-reactive response against the SIVagm Gag p27 protein. Even if responses in AGMs were detected more frequently with the Western blot analyses than with the ELISAs, these responses were different in magnitude and considerably weaker than those in macaques.To compare B- and T-cell responses over time, five simian T-cell leukemia virus-seronegative AGMs were infected with SIVagm.sab92018, and the animals were followed longitudinally during the acute and postacute phases of infection until day 90 postinfection (p.i.). Sequential blood samples were collected and biopsies of auxiliary and inguinal LNs were performed on day −5 and at three times p.i. (days 14, 43, and 62). PVL was measured by real-time PCR (5). Since we searched for Gag p27-specific responses, we also quantified Gag p27 antigen in the plasma (SIV p27 antigen assay; Coulter, Miami, FL). Viral RNA and p27 antigenemia peaks were observed between days 7 and 14 p.i. (Fig. 2A and B, respectively). The Gag p27 levels were variable among the animals but in a range similar to those reported previously in AGMs and macaques (3, 5). As has also been observed in SIVmac infection (except for rapid progressors), plasma Gag p27 levels fell below the detection level in the postacute phase (i.e., after day 28 p.i.) (Fig. ​(Fig.2B2B and data not shown). There were significant increases in circulating CD8⁺ DR⁺ T cells at days 7 and 14 p.i. and in CD8⁺ Ki-67⁺ T cells at days 14 and 28 p.i. (Fig. 2C and D, left panels). After day 28 p.i., the percentages were no longer statistically different from baseline levels. In LN cells (LNCs), the percentage of CD8⁺ Ki-67⁺ T cells rose from 3.1% ± 1.1% before infection to 6.1% ± 0.3% at day 62 p.i., but the difference was not statistically significant (Fig. ​(Fig.2D,2D, right panel). The levels of blood CD4⁺ DR⁺ Ki-67⁺, CD8⁺ DR⁺ Ki-67⁺, CD8⁺ Ki-67⁺ T cells, and LNC CD8⁺ Ki-67⁺ T cells were positively correlated with viremia (P values of 0.002 for DR⁺ cells and P values of <0.02 for Ki-67⁺ cells). Altogether, these results confirm previous data showing early, transient T-cell activation in the peripheral blood of SIVagm-infected AGMs (13).Open in a separate windowFIG. 2.Plasma viremia and T-cell activation in blood and LNs of five longitudinally followed SIVagm.sab₉₂₀₁₈-infected African green monkeys. (A) SIVagm.sab RNA copy numbers in plasma. (B) Plasma Gag p27 concentrations. (C) Percentages of MHC-II DR-positive CD4⁺ (•) and CD8⁺ (○) T cells within, respectively, total CD4⁺ and CD8⁺ T cells from PBMCs and LNCs. (D) Percentages of Ki-67⁺ CD4⁺ (•) and CD8⁺ (○) T cells within, respectively, total CD4⁺ and CD8⁺ T cells from PBMCs and LNCs. Results are shown as the mean ± the standard error of the mean. Asterisks indicate statistically significant differences compared to levels before infection (P < 0.05).We next looked for the presence of Ab responses against rP27 in these animals. No Ab were detected before infection. After infection, all five AGMs developed anti-SIVagm IgGs within 4 to 9 weeks p.i., with AGM 02001 showing the fastest response (Fig. ​(Fig.3A).3A). While the humoral responses against whole virions were significant (Fig. ​(Fig.3B),3B), the anti-rP27 responses were below the threshold for positivity (Fig. ​(Fig.3B),3B), with the exception of one animal (AGM 02001). The anti-rP27 response in this animal was only transient since it was no longer detectable at week 75 p.i., in contrast to the anti-SIV Ab that were sustained (Fig. ​(Fig.3B3B and data not shown).Open in a separate windowFIG. 3.Longitudinal analysis of IgG titers and T-cell proliferative responses against SIVagm and Gag p27 in five AGMs experimentally infected with SIVagm.sab₉₂₀₁₈. (A and B) Ab responses were analyzed by ELISA. (A) IgG dose-response curves against SIVagm (top) and rP27 (bottom) are shown over time (week −1 to week 24 p.i.). O.D.450, optical density at 450 nm. (B) Mid-point titers were calculated as described in the legend to Fig. ​Fig.1A.1A. Continuous lines correspond to median titers from all five animals. Red, anti-SIVagm IgGs; green, anti-p27 IgGs. (C) Proliferative responses of CD4⁺ and CD8⁺ T cells were assessed by flow cytometry using carboxy fluorescein succinimidyl ester staining (CFSE). CD4⁺ and CD8⁺ T-cell responses in PBMCs (left) and LNCs (right) after stimulation with peptide pools (Gag without P27, P27, and Tat) and Gag rP27 are shown for each animal. All data are reported after background subtraction. Results are presented in columns as the mean ± the standard error of the mean. Asterisks indicate statistically significant differences compared to individual values before infection (P < 0.05).We next searched for T-cell responses against Gag p27 compared to other SIVagm antigens in these animals. Gag p27 epitopes were presented in the following two ways: in the context of rP27 and as synthetic peptides. The peptide pools (comprised of overlapping 15-mers) spanned the following SIVagm proteins: Gag p27, Gag without p27, Env, and Tat. The amino acid sequences of the Gag and Env peptides corresponded to the autologous wild-type SIVagm.sab92018 sequence, and those of the Tat peptides corresponded to an SIVagm.sab consensus sequence. The latter was determined using Tat sequences of other SIVagm viruses from Senegal that are available in the databases (SIVagm.sab1c, SIVagm.sabD42, and SIVagm.sabD30). We measured T-cell responses by investigating the antigen-induced proliferation. T cells from blood (peripheral blood mononuclear cells [PBMCs]) and LNs were analyzed. All assays were performed with fresh cells that were stimulated with 10 μg/ml of Gag rP27 and 5 μg/ml of peptides over a period of 4 days. Dead cells were gated out using 7-amino-actinomycin D, and dividing (CFSE^low) cells were analyzed after stimulation with medium alone, SIV antigens, or concanavalin A as a positive control. We detected significant Gag p27-specific proliferative responses for CD8⁺ T cells in PBMCs and for CD4⁺ and CD8⁺ T cells in LNCs (Fig. ​(Fig.3C).3C). The animal with the detectable anti-p27 Ab (AGM 02001) did not show stronger p27-specific T-cell responses than the other animals. Thus, all SIV-infected AGMs were able to mount a proliferative T-cell response against p27, while anti-p27 IgGs were lacking in four of the animals. However, the SIVagm-specific T-cell responses were detected at only a few time points p.i.We then analyzed the T-cell responses in the chronic phase of AGMs naturally and experimentally infected with SIVagm.sab92018. PVL, peripheral blood cell counts (CD4⁺ and CD8⁺ T cells; CD20⁺ B cells), and immune activation (Ki-67⁺ CD4⁺ and CD8⁺ T cells) were similar in naturally infected and in experimentally infected AGMs (Fig. ​(Fig.4A).4A). As expected, cell counts and immune activation levels were also not different from SIV-negative AGMs (Fig. ​(Fig.4A).4A). Again, we measured SIV-specific responses first by a proliferation assay (Fig. ​(Fig.4B).4B). One out of five animals tested had a proliferative SIV-specific CD4⁺ T-cell response (against Gag without p27, P27, rP27, Env GP120, and Tat), and two animals had a CD8⁺ T-cell response (against P27 in both animals and against Env GP120 and Tat in one). Two animals (one naturally infected and one experimentally infected with SIVagm.sab92018) did not show any detectable antigen-specific proliferative CD4⁺ or CD8⁺ T-cell response.Open in a separate windowFIG. 4.Immune parameters and SIVagm-specific proliferative and cytokine T-cell responses in chronically infected AGMs. (A) Cell counts (CD4⁺ and CD8⁺ T cells; B cells) and immune activation levels (percent of Ki-67⁺ in CD4⁺ and CD8⁺ T cells) in AGMs (n = 4) naturally infected with SIVagm (Nat SIV⁺) and AGMs (n = 6) experimentally infected with SIVagm.sab₉₂₀₁₈ (Exp SIV⁺) compared to uninfected AGMs (n = 10) (SIV⁻). PVL, if known, is indicated. Green, blue, and orange symbols correspond, respectively, to noninfected, naturally infected, and experimentally infected AGMs. (B) Proliferative response to SIVagm antigens in chronically infected AGMs (n = 5) compared to those in uninfected AGMs (n = 3). PBMCs were stimulated with the same antigens as those described in the legend to Fig. ​Fig.3.3. (C) Analysis of cytokine responses (gamma interferon [IFN-γ] and tumor necrosis factor alpha [TNF-α]) by SIVagm-specific T cells. ConA was used as a positive control. Representative results from a single animal are shown here. (D) Cumulative values of SIVagm-specific TNF-α and IFN-γ responses in chronically infected animals. The responses to SIVagm antigens were analyzed in peripheral blood specimens of 4 naturally and 5 experimentally infected AGMs as well as 10 uninfected AGMs. The data are reported after background subtraction corresponding to the subtraction of the frequency of positive events from the unstimulated samples to the frequency of positive events from the antigen-specific stimulation. Proliferative T-cell responses and cytokine T-cell responses in SIV-infected AGMs were defined as positive when higher than 3 standard deviations above the mean responses for uninfected animals. Freq, frequency; w/o, without.These results were extended to an analysis of SIV-specific T-cell cytokine responses, e.g., the production of IFN-γ and TNF-α in nine chronically infected compared to 10 noninfected AGMs (Fig. 4C and D). Fresh cells were stimulated for 8 h with the antigens described above. SIV-specific cytokine responses were detected in CD8⁺ but not in CD4⁺ T cells. Seven animals out of nine showed a response against at least one antigen. The two animals showing no response were among the four naturally infected animals tested. We therefore cannot exclude that the absence of response in these two animals is due to the presence of highly divergent viruses. However, a precise epitope mapping in SIVagm sequences would be necessary to confirm this. In those animals showing a SIVagm-specific cytokine T-cell response, the responses were directed against Gag p27 (four out of nine animals), other Gag proteins than p27 (two out of nine animals), and Env GP120 (four out of nine animals). In the experimentally infected animals, we might have underestimated the responses against Tat compared to Gag and Env antigens, since the Tat peptides corresponded to an SIVagm.sab consensus sequence and not to the autologous virus (SIVagm.sab92018). There was no correlation between the magnitude or breadth of SIV-specific T-cell responses and immune activation or PVL.Altogether, our study demonstrates that AGMs can mount T-cell proliferative and cytokine responses against Gag p27. The T-cell response was variable among the animals. In general, it appeared moderate, comparable to chronically SIV-infected RMs (9). Of note, T-cell responses were not consistently detected at all time points and not in all animals. We cannot exclude the possibility that we underestimated the magnitude of the cytokine responses. For instance, we did not costimulate the cells during the assays. However, cytokine responses were also variable in vervet AGMs, with a trend for reduced levels compared to those for RMs, even when more-sensitive assays were used (23). In SM, the responses were also reported to be not stronger than in RMs. This is in line with the lack of efficient control of viral replication in natural hosts (6, 22).In our study, we show that IgG responses against Gag p27 are either lacking, weak, or transient, while Ab against other SIVagm proteins are present. The mechanisms underlying this selective lack of Gag p27 Ab responses are unclear. It could be related to moderate and/or dysfunctional CD4⁺ T-cell responses and/or due to an unknown suppressive regulatory mechanism. SIV-specific T-cell cytokine responses were indeed principally found at the CD8⁺ T-cell level. This was also reported in SIVsm-infected SM (6, 22). Here, we also searched for SIVagm Gag p27-specific proliferative responses. Interestingly, they were detected for CD4⁺ T cells, indicating the presence of p27-specific CD4⁺ memory cells in AGMs. Moreover, AGMs can potentially mount a strong and sustained anti-Gag p27 humoral response, when appropriately immunized (D. Favre et al., unpublished data). This suggests that there is neither a central B-cell tolerance against p27 Gag protein in AGMs nor an inherent inability for CD4⁺ T cells to provide helper B-cell functions. The transient nature of anti-p27 Ab in one animal would be in favor of regulatory mechanisms, but that needs to be confirmed. Another explanation could be that AGMs are able to mount Ab responses against some p27 epitopes but not to those exposed by the native protein, which would explain why we and others detect more frequently humoral responses in Western blot analysis than in ELISAs (16).In conclusion, we characterized the IgG responses against SIVagm and confirmed a lower humoral response against p27 than in RMs. Moreover, our study reveals that cytokine and proliferative T-cell responses against SIVagm Gag p27 are detectable in AGMs. Thus, the reduced ability of the AGM to produce Ab against Gag p27 p.i. is not related to a lack of Gag p27-specific T cells.     

The interaction of beta-amyloid protein with cellular membranes stimulates its own production

Gradual changes in steady-state levels of beta amyloid peptides (Aβ) in brain are considered an initial step in the amyloid cascade hypothesis of Alzheimer's disease. Aβ is a product of the secretase cleavage of amyloid precursor protein (APP). There is evidence that the membrane lipid environment may modulate secretase activity and alters its function. Cleavage of APP strongly depends on membrane properties. Since Aβ perturbs cell membrane fluidity, the cell membrane may be the location where the neurotoxic cascade of Aβ is initiated. Therefore, we tested effects of oligomeric Aβ on membrane fluidity of whole living cells, the impact of exogenous and cellular Aβ on the processing of APP and the role of GM-1 ganglioside. We present evidence that oligoAβ_(1-40) stimulates the amyloidogenic processing of APP by reducing membrane fluidity and complexing with GM-1 ganglioside. This dynamic action of Aβ may start a vicious circle, where endogenous Aβ stimulates its own production. Based on our novel findings, we propose that oligoAβ_(1-40) accelerates the proteolytic cleavage of APP by decreasing membrane fluidity.     

The stability of DNA intrastrand cross-links of antitumor transplatin derivative containing non-bulky methylamine ligands

Oligonucleotides modified by clinically ineffective trans-diamminedichloridoplatinum(II) (transplatin) have been shown to be effective modulators of gene expression. This is so because in some nucleotide sequences the 1,3-GNG intrastrand adducts formed by transplatin in double-helical DNA readily rearrange into interstrand cross-links so that they can cross-link the oligonucleotides to their targets. On the other hand, in a number of other sequences these intrastrand adducts are relatively stable, which represents the major difficulty in the clinical use of the antisense transplatin-modified oligonucleotides. Therefore, we examined in this study, the stability of 1,3-GNG intrastrand adducts in double-helical DNA formed by a new antitumor derivative of transplatin, trans-[Pt(CH₃NH₂)₂Cl₂], in the sequence contexts in which transplatin formed relatively stable intrastrand cross-links which did not readily rearranged into interstrand cross-links. We have found that 1,3-GNG intrastrand adducts in double-helical DNA formed by trans-[Pt(CH₃NH₂)₂Cl₂] even in such sequences readily rearrange into interstrand cross-links. This work also suggests that an enhanced frequency of intrastrand cross-links yielded by trans-[Pt(CH₃NH₂)₂Cl₂] is a consequence of the fact that these DNA lesions considerably distort double-helical DNA in far more sequence contexts than parent transplatin. Our results suggest that trans-[Pt(CH₃NH₂)₂Cl₂]-modified oligonucleotides represent promising candidates for new agents in antisense or antigene approach.     

Transformation of diphenyl ethers by Trametes versicolor and characterization of ring cleavage products

The white-rot fungi Trametes versicolor SBUG 1050, DSM 11269 and DSM 11309 are able to oxidize diphenyl ether and its halogenated derivatives 4-bromo- and 4-chlorodiphenyl ether. The products formed from diphenyl ether were 2- and 4-hydroxydiphenyl ether. Both 4-bromo- and 4-chlorodiphenyl ether were transformed to the corresponding products hydroxylated at the non-halogenated ring. Additionally, ring-cleavage products were detected by high perfomance liquid chromatography and characterized by gas chromatography/mass spectrometry and proton nuclear magnetic resonance spectroscopy. Unhalogenated diphenyl ether was degraded to 2-hydroxy-4-phenoxymuconic acid and 6-carboxy-4-phenoxy-2-pyrone. Brominated derivatives of both these compounds were formed from 4-bromodiphenyl ether, and 4-chlorodiphenyl ether was transformed in the same way to the analogous chlorinated ring cleavage products. Additionally, 4-bromo- and 4-chlorophenol were detected as intermediates from 4-bromo- and 4-chlorodiphenyl ether, respectively. In the presence of the cytochrome-P450 inhibitor 1-aminobenzotriazole, no metabolites were formed by cells of Trametes versicolor from the diphenyl ethers investigated. Cell-free supernatants of whole cultures with high laccase and manganese peroxidase activities were not able to transform the unhydroxylated diphenyl ethers used.