Despite recent evidence of improved graft outcomes and safety, the high incidence of early acute cellular rejection with belatacept, a high-affinity CTLA4-Ig, has limited its use in clinical ...transplantation. Here we define how the incomplete control of endogenous donor-reactive memory T cells results in belatacept-resistant rejection in an experimental model of BALB/c.2W-OVA donor heart transplantation into C57BL/6 recipients presensitized to donor splenocytes. These sensitized mice harbored modestly elevated numbers of endogenous donor-specific memory T cells and alloantibodies compared with naive recipients. Continuous CTLA4-Ig treatment was unexpectedly efficacious at inhibiting endogenous graft-reactive T cell expansion but was unable to inhibit late CD4+ and CD8+ T cell infiltration into the allografts, and rejection was observed in 50% of recipients by day 35 after transplantation. When CTLA4-Ig was combined with the sphingosine 1-phosphate receptor-1 (S1PR1) functional antagonist FTY720, alloantibody production was inhibited and donor-specific IFN-γ-producing T cells were reduced to levels approaching nonsensitized tolerant recipients. Late T cell recruitment into the graft was also restrained, and graft survival improved with this combination therapy. These observations suggest that a rational strategy consisting of inhibiting memory T cell expansion and trafficking into the allograft with CTLA4-Ig and FTY720 can promote allograft survival in allosensitized recipients.
Tuberculosis (TB) is a major health problem requiring an appropriate cell immune response (IR) to be controlled. Since regulatory T cells (Tregs) are relevant in IR regulation, we analyzed Tregs ...variations throughout the course of TB treatment and its relationship with changes in immune-endocrine mediators dealing with disease immunopathology. The cohort was composed of 41 adult patients, 20 of them completing treatment and follow-up. Patients were bled at diagnosis (T0) and at 2 (T2), 4 (T4), 6 (T6), and 9 months following treatment initiation. Twenty-four age- and sex-matched healthy controls (HCo) were also included. Tregs (flow cytometry) from TB patients were increased at T0 (versus HCo P<0.05), showing even higher values at T2 (versus T0 P<0.01) and T4 (versus T0 P<0.001). While IL-6, IFN-γ, TGF-β (ELISA), and Cortisol (electrochemiluminescence, EQ) were augmented, DHEA-S (EQ) levels were diminished at T0 with respect to HCo, with cytokines and Cortisol returning to normal values at T9. Tregs correlated positively with IFN-γ (R=0.868, P<0.05) at T2 and negatively at T4 (R=-0.795, P<0.05). Lowered levels of proinflammatory cytokines together with an increased frequency of Tregs of patients undergoing specific treatment might reflect a downmodulatory effect of these cells on the accompanying inflammation.
Bis-2,3-heteroarylmaleimides and polyheterocondensed imides joined through nitrogen atoms of the N,N′-bis(ethyl)-1,3-propanediamine linker were prepared from substituted maleic anhydrides and ...symmetrical diamines in good to satisfactory yields and short reaction times using microwave heating. The novel molecules were shown to inhibit proliferation of human tumor cells (NCI-H460 lung carcinoma) and rat aortic smooth muscle cells (SMCs) with variable potencies. Compound 11a, the most potent one of the series, showed IC50 values comparable to those observed for the leading molecule elinafide in both cell lines, but with a higher selectivity toward human tumor cells. Compound 11a affected G1/S phase transition of the cell cycle, showed in vitro DNA intercalating activity and in vivo antitumor activity. A thorough structural analysis of the 11a-DNA complex was also made by mean of NMR and computational techniques.
Host-defense peptides (HDPs) are promising compounds against multidrug-resistant microbes. In vitro, their bactericidal and toxic concentrations are significantly different, but this might be due to ...the use of separate assays, with different cell densities. For experiments with a single cell type, the cell-density dependence of the active concentration of the DNS-PMAP23 HDP could be predicted based on the water/cell-membrane partition equilibrium and exhibited a lower bound at low cell counts. On the basis of these data, in the simultaneous presence of both bacteria and an excess of human cells, one would expect no significant toxicity, but also inhibition of the bactericidal activity due to peptide sequestration by host cells. However, this inhibition did not take place in assays with mixed cell populations, showing that for the HDP esculentin-1a(1–21)NH2, a range of bactericidal, nontoxic concentrations exists and confirming the effective selectivity of HDPs. Mixed-cell assays might be necessary to effectively asses HDP selectivity.
OBJECTIVEClinic blood pressure values are known to change according to seasonal influences. We therefore examined home and 24 h ambulatory blood pressure values to determine whether these ...measurements are also affected by the seasons.
DESIGN AND METHODSIn 2051 subjects of the Pressione Arteriose Monitorate E Loro Associazioni (PAMELA) study, we measured clinic (sphygmomanometric measurements), home (semi-automatic device) and ambulatory (Spacelabs 90207) systolic blood pressure, diastolic blood pressure and heart rate. Because the overall sample was evenly distributed over each month (except August), we were able to make a cross-sectional determination of whether the values differed between seasons. The corresponding heart rates were also evaluated.
RESULTSAs expected, summer was associated with the lowest clinic blood pressure and winter with the highest, and this was the case also for home and 24 h average blood pressure, although seasonal differences in the latter were less pronounced. Seasonal clinic, home and ambulatory blood pressure patterns were similar for normotensive subjects (n = 1152), untreated hypertensives (n = 540) and treated hypertensives (n = 359). Heart rate values did not differ by season.
CONCLUSIONSSeasonal influences on blood pressure are not limited to conventional measurements but characterize daily values as well. These effects are visible in both normal and elevated blood pressure values, regardless of the effect of antihypertensive drugs. This has implications both for the clinician and for studies aimed at evaluating the effects of antihypertensive treatment.
Background
Clinicopathological research has focused on identifying molecular and biological prognostic factors for laryngeal carcinoma (LSCC) treated with post‐operative radiotherapy (RT). The aim of ...this study was to assess the prognostic importance of anti‐apoptotic proteins survivin and B‐cell lymphoma‐2 (Bcl‐2) in a series of patients with LSCC who had primary surgery followed by RT.
Methods
Thirty‐three consecutive patients who underwent primary surgery followed by RT were considered. Survivin nuclear and cytoplasmic expressions and Bcl‐2 expression were determined immunohistochemically.
Results
The loco‐regional recurrence rate was significantly higher among LSCC patients with a nuclear survivin expression >10.0% (P = 0.029), and their disease‐free survival (DFS) was shorter than in cases whose nuclear survivin expression was ≤10.0% (P = 0.002). DFS was significantly shorter in cases with a Bcl‐2 expression >2.0% than in those whose Bcl‐2 expression was ≤2.0% (P = 0.035).
Conclusions
Nuclear survivin expression and Bcl‐2 expression warrant further investigation as potential predictive biomarkers to enable individualized treatments (e.g. post‐operative chemo‐radiotherapy instead of RT alone for patients whose LSCCs strongly express nuclear survivin or/and Bcl‐2). This preliminary evidence justifies the design of new studies on the association of agents targeting survivin and Bcl‐2 with conventional chemotherapeutic agents and RT for advanced LSCC.
A series of 2,3-heteroarylmaleimides and polyheterocondensed imides were prepared and their antiproliferative activity was tested against human tumor cells (NCI-H460 lung carcinoma) and rat SMCs.
A ...series of 2,3-heteroarylmaleimides
9 and polyheterocondensed imides
12 were prepared in good yields and short reaction time using a very efficient procedure consisting in the condensation of the corresponding anhydrides and
N,
N-diethylethylenediamine and microwave heating. The antiproliferative activity of the novel molecules was tested against human tumor cells (NCI-H460 lung carcinoma) and rat aortic smooth muscle cells (SMCs). The IC
50 values for the novel molecules ranged from 0.08 to 13.9
μM in SMCs, and from 0.84 to 9
μM in the tumor cell line. The activity profile for compounds
9 and
12 is comparable to that obtained for amonafide in NCI-H460, except for fused imides
12b,
i which proved to be about 10-fold more potent. Whereas, in rat SMCs, only the compound
12b was shown to be 10-fold more potent than amonafide. Instead
12c is equipotent to amonafide. These results suggest that the extended π-system and the kind of heteroatom are essential in the binding with the molecular target.
Antimicrobial peptides (AMPs) represent new alternatives to cope with the increasing number of multi-drug resistant microbial infections. Recently, a derivative of the frog-skin AMP esculentin-1a, ...Esc(1-21), was found to rapidly kill both the planktonic and biofilm forms of the Gram-negative bacterium Pseudomonas aeruginosa with a membrane-perturbing activity as a plausible mode of action. Lately, its diastereomer Esc(1-21)-1c containing two d-amino acids i.e. DLeu14 and DSer17 revealed to be less cytotoxic, more stable to proteolytic degradation and more efficient in eradicating Pseudomonas biofilm. When tested in vitro against the free-living form of this pathogen, it displayed potent bactericidal activity, but this was weaker than that of the all-l peptide. To investigate the reason accounting for this difference, mechanistic studies were performed on Pseudomonas spheroplasts and anionic or zwitterionic membranes, mimicking the composition of microbial and mammalian membranes, respectively. Furthermore, structural studies by means of optical and nuclear magnetic resonance spectroscopies were carried out. Our results suggest that the different extent in the bactericidal activity between the two isomers is principally due to differences in their interaction with the bacterial cell wall components. Indeed, the lower ability in binding and perturbing anionic phospholipid bilayers for Esc(1-21)-1c contributes only in a small part to this difference, while the final effect of membrane thinning once the peptide is inserted into the membrane is identical to that provoked by Esc(1-21). In addition, the presence of two d-amino acids is sufficient to reduce the α-helical content of the peptide, in parallel with its lower cytotoxicity.
Effect of d-amino acid incorporation in Esc(1-21) on the bacterial killing. Display omitted
•The presence of DLeu14 and DSer17 in Esc(1-21) reduces α-helix and cytotoxicity.•Both isomers have similar effects on model membranes and spheroplasts.•The different activity involves interaction with the bacterial cell wall.
Antimicrobial peptides (AMPs) represent a promising class of compounds to fight resistant infections. They are commonly thought to kill bacteria by perturbing the permeability of their cell ...membranes. However, bacterial killing requires a high coverage of the cell surface by bound peptides, at least in the case of cationic and amphipathic AMPs. Therefore, it is conceivable that peptide accumulation on the bacterial membranes might interfere with vital cellular functions also by perturbing bilayer dynamics, a hypothesis that has been termed “sand in the gearbox”. Here we performed a systematic study of such possible effects, for two representative peptides (the cationic cathelicidin PMAP-23 and the peptaibol alamethicin), employing fluorescence and NMR spectroscopies. These approaches are commonly applied to characterize lipid order and dynamics, but sample different time-scales and could thus report on different membrane properties. In our case, fluorescence anisotropy measurements on liposomes labelled with probes localized at different depths in the bilayer showed that both peptides perturb membrane fluidity and order. Pyrene excimer-formation experiments showed a peptide-induced reduction in lipid lateral mobility. Finally, laurdan fluorescence indicated that peptide binding reduces water penetration below the headgroups region. Comparable effects were observed also in fluorescence experiments performed directly on live bacterial cells. By contrast, the fatty acyl chain order parameters detected by deuterium NMR spectroscopy remained virtually unaffected by addition of the peptides. The apparent discrepancy between the two techniques confirms previous sporadic observations and is discussed in terms of the different characteristic times of the two approaches. The perturbation of membrane dynamics in the ns timescale, indicated by the multiple fluorescence approaches reported here, could contribute to the antimicrobial activity of AMPs, by affecting the function of membrane proteins, which is strongly dependent on the physicochemical properties of the bilayer.
Display omitted
•Perturbation of lipid dynamics by different antimicrobial peptides was characterized.•Fluorescence experiments show that membrane dynamics is hindered.•Solid state NMR indicates that they cause a decrease in lipid order or no effect.•The inconsistency between these methods is likely due to their different timescales•Perturbation of membrane dynamics in the ns could contribute to bacterial killing.
Abstract
Establishing a state of transplantation tolerance has been achieved in limited numbers of transplant recipients in the clinic. Clinical observations suggest that donor-specific antibodies ...can mediate allograft rejection, leading us to hypothesize that stable transplantation tolerance requires donor-specific B cell responses to also be suppressed. We hypothesize that this can be achieved through the induction of B cell-intrinsic tolerance. Using a well-established experimental model of transplantation tolerance induced to allogeneic B/c hearts with anti-CD154+donor spleen cell transfusion, we observed that donor-specific B cells are intrinsically tolerant. B cells from tolerant recipients did not produce anti-B/c IgG when adoptively transferred into naïve MD4 host and challenged with B/c splenocytes or heart grafts, and were blocked in their ability to differentiate into germinal center (GC) B cells. To test whether tolerant B cells could be rescued by ongoing GC responses, we adoptively transferred tolerant B cells into congenic hosts, followed by B/c spleen cell immunization. Tolerant B cells did not recover their ability to differentiate into antibody secreting cells, but instead they are able to suppress IgG production by naïve host B cells. Finally, we show that donor-specific tolerant B cells are not able to suppress third-party antibody responses nor exhibit linked suppression. Taken together, our data demonstrate that tolerant donor-specific B cells are profoundly altered compared to naïve B cells: they have significantly diminished ability to differentiate into antibody secreting cells, but instead acquired the ability to suppress donor-specific, but not third-party, antibody responses by naïve B cells.
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