Protective effect of immunization with nonviral antigens against Friend leukemia virus in mice

DBA/2 mice immunized with poly(A).poly(U) complexed with methylated bovine serum albumin and emulsified in Freund's complete adjuvant were protected against challenge with Friend leukemia virus. There was no correlation between the level of antibody to the immunogen in the prechallenge serum and induced resistance to the virus. Although prechallenge sera of mice given the same amount of the duplex in a single inoculum bound 9.7% of poly(A).poly([(3)H]U) input, as compared to 45.3% bound by the prechallenge sera of mice given the immunogen in divided doses, both groups of mice were equally resistant to infection. Immunization with two other nonviral agents, bovine serum albumin fraction V or dinitrophenylated keyhole limpet hemocyanin, induced the same level of protection. A sparing effect of approximately 10(1.5) in infectivity was afforded the immunized mice. Immunization with either poly(A).poly(U) alone or with the carrier methylated bovine serum albumin was ineffective.In addition to antibodies to the respective immunogens, the prechallenge sera of the immunized mice also contained antibody to Friend leukemia virus gp71. The presence of such viral antibodies was not always related to resistance to infection by Friend virus. Some immunized mice that survived infection did not have gp71 antibody in their serum before challenge, and mice immunized with poly(A).poly(U) alone were susceptible to infection, although their prechallenge sera contained antibody to gp71. The mechanism involved in the induction of resistance to infection is not known. The effect may be mediated through a modification of the expression of both endogenous and exogenous type C viruses and affect immunological mechanisms controlling cellular responses.     

XerD-mediated FtsK-independent integration of TLC? into the Vibrio cholerae genome

As in most bacteria, topological problems arising from the circularity of the two Vibrio cholerae chromosomes, chrI and chrII, are resolved by the addition of a crossover at a specific site of each chromosome, dif, by two tyrosine recombinases, XerC and XerD. The reaction is under the control of a cell division protein, FtsK, which activates the formation of a Holliday Junction (HJ) intermediate by XerD catalysis that is resolved into product by XerC catalysis. Many plasmids and phages exploit Xer recombination for dimer resolution and for integration, respectively. In all cases so far described, they rely on an alternative recombination pathway in which XerC catalyzes the formation of a HJ independently of FtsK. This is notably the case for CTXϕ, the cholera toxin phage. Here, we show that in contrast, integration of TLCϕ, a toxin-linked cryptic satellite phage that is almost always found integrated at the chrI dif site before CTXϕ, depends on the formation of a HJ by XerD catalysis, which is then resolved by XerC catalysis. The reaction nevertheless escapes the normal cellular control exerted by FtsK on XerD. In addition, we show that the same reaction promotes the excision of TLCϕ, along with any CTXϕ copy present between dif and its left attachment site, providing a plausible mechanism for how chrI CTXϕ copies can be eliminated, as occurred in the second wave of the current cholera pandemic.The causative agent of the epidemic severe diarrheal disease cholera is the Vibrio cholerae bacterium. A major determinant of its pathogenicity, the cholera enterotoxin, is encoded in the genome of the filamentous cholera toxin phage, CTXϕ (1). Like many other V. cholerae filamentous phages, CTXϕ uses a host chromosomally encoded, site-specific recombination (Xer) machinery for lysogenic conversion (2–4). The Xer machinery normally serves to resolve chromosome dimers, which result from homologous recombination events between the two chromatids of circular chromosomes during or after replication. In V. cholerae, as in most bacteria, the Xer machinery consists of two tyrosine recombinases, XerC and XerD. They act at a unique specific chromosomal site, dif, on each of the two circular chromosomes, chrI and chrII, of the bacterium (5). Integrative mobile elements exploiting Xer (IMEXs) carry a dif-like site on their circular genome, attP (3, 4) (Fig. 1A). XerC and XerD promote their integration by catalyzing a recombination event between this site and a cognate chromosomal dif site (3, 4) (Fig. 1A). Based on the structure of their attP site, IMEXs can be grouped into at least three families (3, 4) (Fig. 1B). In all cases, however, a new functional dif site is restored after integration, which permits multiple successive integration events (Fig. 1A). Indeed, most clinical and environmental V. cholerae isolates harbor large IMEX arrays (6, 7).Open in a separate windowFig. 1.Systems that use Xer. (A) Scheme depicting the sequential integration of IMEXs. Triangles represent attP and dif sites, pointing from the XerD binding site to the XerC binding site. Chromosomal DNA (black), TLCϕ DNA (blue), and CTXϕ DNA (magenta) are indicated. Dotted triangles represent nonfunctional CTXϕ sites. (B) Sequence alignment of dif1, attP^CTX, attP^VGJ, attP^TLC, difA, and dif2. Bases differing from dif1 are indicated in color. Bases that do not fit the XerD binding site consensus are indicated in lowercase. XerC (●) and XerD (○) cleavage points are indicated. (C) Xer recombination pathways. XerC (light gray circles), XerD (dark gray circles), dif sites (red and black lines), and attP^CTX and attP^VGJ (magenta and green lines) are indicated. XerC and XerD catalysis-suitable conformations are depicted as horizontal and vertical synapses, respectively. Cleavage points are indicated as in B.IMEX array formation participates in the continuous and rapid dissemination of new cholera toxin variants in at least three ways. First, CTXϕ integration is intrinsically irreversible because the active form of its attP site consists of the stem of a hairpin of its ssDNA genome, which is masked in the host dsDNA genome (8, 9) (Fig. 1 A and B). However, free CTXϕ genome copies can be produced by a process analogous to rolling circle replication after the integration of a second IMEX harboring the same integration/replication machinery, such as the RS1 satellite phage, which permits the production of new CTXϕ viral particles (10). Second, the V. cholerae Gillermo Javier filamentous phage (VGJϕ) belongs to a second category of IMEXs whose attP site permits cycles of integration and excision by Xer recombination (11). VGJϕ excision allows for the formation of hybrid molecules harboring the concatenated genomes of CTXϕ and VGJϕ, provided that VGJϕ integrated before CTXϕ (11). The hybrid molecules can be packaged into VGJϕ particles. VGJϕ particles have a different receptor than CTXϕ, which permits transduction of the cholera toxin genes to cells that do not express the receptor of CTXϕ (11–13). Finally, integration of the toxin-linked cryptic phage (TLCϕ), a satellite phage that defines a third category of IMEXs, seems to be a prerequisite to the toxigenic conversion of many V. cholerae strains (14, 15). IMEXs from this family are found integrated in the genome of many bacteria outside of the Vibrios, including human, animal, and plant pathogens, which sparked considerable interest in the understanding of how they exploit the Xer machinery at the molecular level (3, 4).Xer recombination sites consist of 11-bp XerC and XerD binding arms, separated by an overlap region at the border of which recombination occurs (Fig. 1B). XerC and XerD each promote the exchange of a specific pair of strands (Fig. 1B). Recombination between dif sites is under the control of a cell division protein, FtsK, which restricts it temporally to the time of constriction and spatially to a specific zone within the terminus region of chromosomes (16–19). FtsK triggers the formation of a Holliday junction (HJ) by XerD catalysis, which is converted into product by XerC catalysis after isomerization (20, 21) (Fig. 1C). The intermediate HJ is stable enough to be converted into product by replication when XerC catalysis is impeded (5, 17) (Fig. 1C). The integration of IMEXs of the CTXϕ and VGJϕ families escapes FtsK control. The lack of homology in the overlap regions of their attP sites and the dif sites they target prevents any potential XerD-mediated strand exchange (Fig. 1B). CTXϕ and VGJϕ rely on the exchange of a single pair of strands by XerC catalysis for integration, with the resulting HJ being converted into product by replication (8, 9, 11) (Fig. 1C). In the case of CTXϕ, integration is facilitated by an additional host factor, EndoIII, which impedes futile cycles of XerC catalysis once the pseudo-HJ is formed (22) (Fig. 1C). In contrast, the overlap region of TLCϕ attP, attP^TLC, is fully homologous to the overlaps of dif1 and difA, the two sites in which it was found to be integrated (Fig. 1B). Four integration pathways could thus be considered, depending on whether recombination is initiated by XerC or XerD catalysis, and whether it ends with a second pair of strand exchange or not. In addition, attP^TLC lacks a consensus XerD binding site, which could affect the whole recombination process (Fig. 1B).Here, we show that attP^TLC is a poor XerD binding substrate. Nevertheless, we show that TLCϕ integration is initiated by XerD catalysis and that the resulting HJ is converted into product by XerC catalysis. We further show that TLCϕ integration is independent of FtsK. Finally, we demonstrate that the same reaction can lead to the excision of TLCϕ–CTXϕ arrays, providing a plausible mechanism for how all of the CTXϕ copies integrated on V. cholerae chrI can be eliminated in a single step, as occurred in ancestors of strains from the second wave of the current cholera pandemic (23–25).     

Dacarbazine alone or associated with melanoma-bearing cancer pain model induces painful hypersensitivity by TRPA1 activation in mice

Antineoplastic therapy has been associated with pain syndrome development characterized by acute and chronic pain. The chemotherapeutic agent dacarbazine, used mainly to treat metastatic melanoma, is reported to cause painful symptoms, compromising patient quality of life. Evidence has proposed that transient receptor potential ankyrin 1 (TRPA1) plays a critical role in chemotherapy-induced pain syndrome. Here, we investigated whether dacarbazine causes painful hypersensitivity in naive or melanoma-bearing mice and the involvement of TRPA1 in these models. Mouse dorsal root ganglion (DRG) neurons and human TRPA1-transfected HEK293 (hTRPA1-HEK293) cells were used to evaluate the TRPA1-mediated calcium response evoked by dacarbazine. Mechanical and cold allodynia were evaluated after acute or repeated dacarbazine administration in naive mice or after inoculation of B16-F10 melanoma cells in C57BL/6 mice. TRPA1 involvement was investigated by using pharmacological and genetic tools (selective antagonist or antisense oligonucleotide treatment and Trpa1 knockout mice). Dacarbazine directly activated TRPA1 in hTRPA1-HEK293 cells and mouse DRG neurons and appears to sensitize TRPA1 indirectly by generating oxidative stress products. Moreover, dacarbazine caused mechanical and cold allodynia in naive but not Trpa1 knockout mice. Also, dacarbazine-induced nociception was reduced by the pharmacological TRPA1 blockade (antagonism), antioxidants, and by ablation of TRPA1 expression. TRPA1 pharmacological blockade also reduced dacarbazine-induced nociception in a tumor-associated pain model. Thus, dacarbazine causes nociception by TRPA1 activation, indicating that this receptor may represent a pharmacological target for treating chemotherapy-induced pain syndrome in cancer patients submitted to antineoplastic treatment with dacarbazine.     

Facing successfully high mental workload and stressors: An fMRI study

The present fMRI study aimed at highlighting patterns of brain activations and autonomic activity when confronted with high mental workload and the threat of auditory stressors. Twenty participants performed a complex cognitive task in either safe or aversive conditions. Our results showed that increased mental workload induced recruitment of the lateral frontoparietal executive control network (ECN), along with disengagement of medial prefrontal and posterior cingulate regions of the default mode network (DMN). Mental workload also elicited an increase in heart rate and pupil diameter. Task performance did not decrease under the threat of stressors, most likely due to efficient inhibition of auditory regions, as reflected by a large decrement of activity in the superior temporal gyri. The threat of stressors was also accompanied with deactivations of limbic regions of the salience network (SN), possibly reflecting emotional regulation mechanisms through control from dorsal medial prefrontal and parietal regions, as indicated by functional connectivity analyses. Meanwhile, the threat of stressors induced enhanced ECN activity, likely for improved attentional and cognitive processes toward the task, as suggested by increased lateral prefrontal and parietal activations. These fMRI results suggest that measuring the balance between ECN, SN, and DMN recruitment could be used for objective mental state assessment. In this sense, an extra recruitment of task‐related regions and a high ratio of lateral versus medial prefrontal activity may represent a relevant marker of increased but efficient mental effort, while the opposite may indicate a disengagement from the task due to mental overload and/or stressors.     

High dose daily interferon-alpha induction and secondary adjunction of ribavirin in treatment-naive patients with chronic hepatitis C. A multicentric, randomised, controlled trial

OBJECTIVES: To evaluate in naive patients with chronic hepatitis C 1- the efficacy and safety of one month interferon alpha (IFN-alpha) induction regimen; 2- the potential virological benefit of a secondary adjunction of ribavirin among HCV RNA negative patients after 20 weeks of IFN therapy, with or without an initial 4-week IFN induction. MATERIAL AND METHODS: 151 naive HCV-RNA positive patients presenting with biopsy- proven chronic hepatitis C and elevated ALT were randomised in a 2: 1 ratio in two arms: IFN-alpha 3 MU thrice a week (tiw) for 24 weeks (non-induced patients); IFN-alpha 6 MU daily for two weeks, then 3 MU daily for two weeks then 3 MU tiw for 20 weeks (induced patients). At week 24, HCV-RNA negative patients were randomised to receive in addition or not ribavirin 1-1.2 g daily for 24 additional weeks. Induction efficacy was assessed on the early viral response (EVR) defined as undetectable HCV RNA at week 4 then week 20. Ribavirin efficacy was assessed on the proportion of maintained complete response until the end of follow-up, 24 weeks after discontinuation of treatment. Data were analysed on an intent-to-treat basis. RESULTS: Efficacy of IFN-alpha induction: 104 patients were randomised to the non-induction group, 47 to the induction group. Gender, age, genotype distribution and HCV viral load at baseline did not differ significantly between the two groups. There was one treatment discontinuation because of adverse events in induced patients versus four in non-induced patients (P > 0.05). The 4 week EVR was significantly greater in induced patients in patients with HCV genotype 1, 4 or 5 (47% vs 12%, P=0.0002) only. There was no impact of induction in patients with HCV genotype 2 or 3. Efficacy of ribavirin: at week 24, 28 and 26 HCV-RNA negative patients were randomised to addition of ribavirin or not, respectively. Patients randomised to secondary additive ribavirin were more often HCV-RNA negative at the end of follow-up than patients treated with IFN-alpha alone: 18/28 (64%) vs 10/26 (39%); P=0.06. Among patients randomised to bitherapy, the relapse rate was significantly lower in patients with genotype 2 or 3 (0/12 vs 6/13, P=0.01) and not in those with genotype 1, 4 or 5 (5/11 vs 3/6, P=0.99). CONCLUSION: A 4 week IFN-alpha induction significantly increases the EVR rate in patients with HCV genotype 1, 4 or 5. Late secondary adjunction of ribavirin to IFN-alpha for 6 months in HCV-RNA negative patients after 6 months of IFN-alpha significantly decreases the relapse rate in patients with HCV genotype 2 or 3, but not in patients with genotypes 1, 4 or 5.     

Analysis of susceptibility of NOD mice to spontaneous and experimentally induced thyroiditis

Beside diabetes, non-obese diabetic (NOD) mice develop sporadic lymphoid infiltration of the thyroid gland, mimicking Hashimoto's thyroiditis. We have examined the prevalence of those manifestations in NOD mice, the influence of the major histocompatibility complex (MHC) and the association with autoantibodies. The incidence at 1 year is of 14.3% in wild-type NOD mice versus 19.6% in congenic NOD.H2^k mice. The moderate, but statistically significant difference, based on the analysis of 161 NOD and 169 NOD.H2^k mice, suggests that MHC genes partially control spontaneous NOD thyroiditis. Autoantibodies against thyroglobulin (Tg) are mouse specific and their presence correlates closely with thyroiditis. The strong correlation between cellular and humoral anomalies therefore resembles Hashimoto's thyroiditis. NOD and NOD.H2^k mice actively immunized against Tg develop severe chronic lesions with epithelium necrosis and interstitial tissue fibrosis. Most interestingly, those lesions do not regress spontaneously as in CBA/J mice. Paradoxically, the response to Tg of lymph node cells from NOD mice is weaker both in proliferation and cytokine production. The defect is most evident for interferon-γ-producing T cells and is reflected in the marked deficit in IgG2a antibodies. Thus a moderate anti-Tg response seems to favor chronicity of thyroiditis. In conclusion, NOD and NOD.H2^k mice offer a unique opportunity of analyzing the factors leading to immune chronicity in a genetic context which promotes autoimmune endocrinopathies.     

A subset of dendritic cells induces CD4+ T cells to produce IFN-gamma by an IL-12-independent but CD70-dependent mechanism in vivo

Interferon (IFN)-gamma, a cytokine critical for resistance to infection and tumors, is produced by CD4(+) helper T lymphocytes after stimulation by cultured dendritic cells (DCs) that secrete a cofactor, interleukin (IL)-12. We have identified a major IL-12-independent pathway whereby DCs induce IFN-gamma-secreting T helper (Th)1 CD4(+) T cells in vivo. This pathway requires the membrane-associated tumor necrosis family member CD70 and was identified by targeting the LACK antigen from Leishmania major within an antibody to CD205 (DEC-205), an uptake receptor on a subset of DCs. Another major DC subset, targeted with 33D1 anti-DCIR2 antibody, also induced IFN-gamma in vivo but required IL-12, not CD70. Isolated CD205(+) DCs expressed cell surface CD70 when presenting antigen to T cell receptor transgenic T cells, and this distinction was independent of maturation stimuli. CD70 was also essential for CD205(+) DC function in vivo. Detection of the IL-12-independent IFN-gamma pathway was obscured with nontargeted LACK, which was presented by both DC subsets. This in situ analysis points to CD70 as a decision maker for Th1 differentiation by CD205(+) DCs, even in Th2-prone BALB/c animals and potentially in vaccine design. The results indicate that two DC subsets have innate propensities to differentially affect the Th1/Th2 balance in vivo and by distinct mechanisms.