Purpose: High-risk human papillomavirus (HPV) is the main etiologic factor for cervical cancer.The severity of HPV-associated cervical lesions has been correlated to the number of infiltrating macrophages. The objective of this work is to characterize the role of tumor-associated macrophages (TAM) on the immune cellular response against the tumor. Experimental Design: We used the HPV16 E6-and E7-expressingTC-1mouse tumor model to study the effect ofTAM onT-cell function in vitro, and depletedTAM, using clodronate-containing liposomes, to characterize its role in vivo. Results: TAM, characterized by the positive expression of CD45, F4/80, and CD11b, formed the major population of infiltrating tumor cells. TAM displayed high basal Arginase I activity, producing interleukin-10 (IL-10); they were resistant to iNOSII activity induction, therefore reversion to M1 phenotype, when stimulated in vitro with lipopolysaccharide/IFNg, indicating an M2 phentoype. In cultures of isolated TAM, TAM induced regulatory phenotype, characterized by IL-10 and Foxp3 expression, and inhibited proliferation of CD8 lymphocytes. In vivo, depletion of TAM inhibited tumor growth and stimulated the infiltration of tumors by HPV16 E7 49-57 -specific CD8 lymphocytes, whereas depletion of Gr1 + tumor-associated cells had no effect. Conclusions: M2-like macrophages infiltrate HPV16-associated tumors causing suppression of antitumorT-cell response, thus facilitating tumor growth. Depletion or phenotype alteration of this population should be considered in immunotherapy strategies.

The cecropin-melittin hybrid antimicrobial peptide BP100 (H-KKLFKKILKYL-NH2) is selective for Gram-negative bacteria, negatively charged membranes, and weakly hemolytic. We studied BP100 conformational and functional properties upon interaction with large unilamellar vesicles, LUVs, and giant unilamellar vesicles, GUVs, containing variable proportions of phosphatidylcholine (PC) and negatively charged phosphatidylglycerol (PG). CD and NMR spectra showed that upon binding to PG-containing LUVs BP100 acquires α-helical conformation, the helix spanning residues 3-11. Theoretical analyses indicated that the helix is amphipathic and surface-seeking. CD and dynamic light scattering data evinced peptide and/or vesicle aggregation, modulated by peptide:lipid ratio and PG content. BP100 decreased the absolute value of the zeta potential (ζ) of LUVs with low PG contents; for higher PG, binding was analyzed as an ion-exchange process. At high salt, BP100-induced LUVS leakage requires higher peptide concentration, indicating that both electrostatic and hydrophobic interactions contribute to peptide binding. While a gradual release took place at low peptide:lipid ratios, instantaneous loss occurred at high ratios, suggesting vesicle disruption. Optical microscopy of GUVs confirmed BP100-promoted disruption of negatively charged membranes. The mechanism of action of BP100 is determined by both peptide:lipid ratio and negatively charged lipid content. While gradual release results from membrane perturbation by a small number of peptide molecules giving rise to changes in acyl chain packing, lipid clustering (leading to membrane defects), and/or membrane thinning, membrane disruption results from a sequence of events - large-scale peptide and lipid clustering, giving rise to peptide-lipid patches that eventually would leave the membrane in a carpet-like mechanism.

Gomesin is a potent cationic antimicrobial peptide (z = +6) isolated from the Brazilian spider Acanthoscurria gomesiana . The interaction of gomesin with large unilamellar vesicles composed of a 1:1 mixture of zwitterionic (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and anionic (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) phospholipids is studied with isothermal titration calorimetry (ITC). In parallel, light scattering and optical microscopy are used to assess peptide-induced vesicle aggregation. The ability of gomesin to permeabilize the membrane is examined with fluorescence spectroscopy of the leakage of 5,6-carboxyfluorescein (CF). Vesicles coated with 3 mol % 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PE-PEG) lipids are also investigated to assess the influence of peptide-induced vesicle aggregation in the activity of gomesin. The ITC and light scattering titrations are done in two ways: lipid into peptide and peptide into lipid injections. Although some differences arise between the two setups, the basic interaction of gomesin with anionic vesicles is preserved. A surface partition model combined with the Gouy-Chapman theory is put forward to fit the ITC results. The intrinsic binding constant of gomesin is found to be K ≈ 10(3) M(-1). The interaction of gomesin with anionic membranes is highly exothermic and enthalpy-driven. Binding of gomesin is virtually always accompanied by vesicle aggregation and changes in membrane permeability, leading to CF leakage. Addition of PE-PEG to the membrane strongly attenuates vesicle aggregation but does not significantly change the mode of action of gomesin. The results point to a strong interaction of gomesin with the membrane surface, causing membrane rupture without a deep penetration into the bilayer core.

No abstract

Inflammation contributes to the pathogenesis of chronic kidney disease (CKD). Molecules released by the inflamed injured tissue can activate toll-like receptors (TLRs), thereby modulating macrophage and CD4(+) T-cell activity. We propose that in renal fibrogenesis, M2 macrophages are recruited and activated in a T helper subset 2 cell (T(H)2)-prone inflammatory milieu in a MyD88-dependent manner. Mice submitted to unilateral ureteral ligation (UUO) demonstrated an increase in macrophage infiltration with collagen deposition after 7 d. Conversely, TLR2, TLR4 and MyD88 knockout (KO) mice had an improved renal function together with diminished T(H)2 cytokine production and decreased fibrosis formation. Moreover, TLR2, TLR4 and MyD88 KO animals exhibited less M2 macrophage infiltration, namely interleukin (IL)-10(+) and CD206(+) CD11b(high) cells, at 7 d after surgery. We evaluated the role of a T(H)2 cytokine in this context, and observed that the absence of IL-4 was associated with better renal function, decreased IL-13 and TGF-β levels, reduced arginase activity and a decrease in fibrosis formation when compared with IL-12 KO and wild-type (WT) animals. Indeed, the better renal outcomes and the decreased fibrosis formation were restricted to the deficiency of IL-4 in the hematopoietic compartment. Finally, macrophage depletion, rather than the absence of T cells, led to reduced lesions of the glomerular filtration barrier and decreased collagen deposition. These results provide evidence that future therapeutic strategies against renal fibrosis should be accompanied by the modulation of the M1:M2 and T(H)1:T(H)2 balance, as T(H)2 and M2 cells are predictive of fibrosis toward mechanisms that are sensed by innate immune response and triggered in a MyD88-dependent pathway.

The need for reliable, fast diagnostics is closely linked to the need for safe, effective treatment of the so-called “neglected” diseases. The list of diseases with no field-adapted diagnostic tools includes leishmaniasis, shigella, typhoid, and bacterial meningitis. Leishmaniasis, in particular, is a parasitic disease caused by Leishmania spp. transmitted by infected phlebotomine sandfly, which remains a public health concern in developing countries with ca. 12 million people infected and 350 million at risk of infection. Despite several attempts, methods for diagnosis are still noneffective, especially with regard to specificity due to false positives with Chagas’ disease caused by Trypanosoma cruzi. Accepted golden standards for detecting leishmaniasis involve isolation of parasites either microscopically, or by culture, and in both methods specimens are obtained by invasive means. Here, we show that efficient distinction between cutaneous leishmaniasis and Chagas’ disease can be obtained with a low-cost biosensor system made with nanostructured films containing specific Leishmania amazonensis and T. cruzi antigens and employing impedance spectroscopy as the detection method. This unprecedented selectivity was afforded by antigen−antibody molecular recognition processes inherent in the detection with the immobilized antigens, and by statistically correlating the electrical impedance data, which allowed distinction between real samples that tested positive for Chagas’ disease and leishmaniasis. Distinction could be made of blood serum samples containing 10−5 mg/mL of the antibody solution in a few minutes. The methods used here are generic and can be extended to any type of biosensor, which is important for an effective diagnosis of many other diseases.

No abstract

Antimicrobial peptides are part of the innate immune system of animals and plants. Their lytic activity against microorganisms generally depends on their ability to disrupt and permeabilize membranes. Here we study the structure-activity relationship of the antimicrobial peptide gomesin (Gm), from the spider Acanthoscurria gomesiana, with large unilamellar vesicles (LUVs) composed of 3:7 palmitoyloleoyl phosphatidylglycerol: palmitoyloleoyl phosphatidylcholine. Several synthetic analogues of Gm were designed to alter the hydrophobicity/charge of the molecule, whereby selected amino acid residues were replaced by alanine. Isothermal titration calorimetry (ITC) was used to assess the thermodynamic parameters of peptide binding to LUVs and light scattering measurements were made to evaluated peptide-induced vesicle aggregation. The ability of the peptides to permeabilize vesicles was quantified through the leakage of an entrapped fluorescent probe. The activity of peptides could be quantified in terms of the leakage extent induced and their affinity to the membrane, which was largely dictated by the exothermic enthalpy change. The results show that analogues more hydrophobic than Gm display higher activity, whereas peptides more hydrophilic than Gm have their activity almost abolished. Vesicle aggregation, on the other hand, largely increases with peptide charge. We conclude that interaction of Gm with membranes depends on an interplay between surface electrostatic interactions, which drive anchoring to the membrane surface and vesicle aggregation, and insertion of the hydrophobic portion into the membrane core, responsible for causing membrane rupture/permeabilization.