The synergy between structural stability and DNA-binding controls the antibody production in EPC/DOTAP/DOPE liposomes and DOTAP/DOPE lipoplexes

We present a comparative study of the physico-chemical properties, in vitro cytotoxicity and in vivo antibody production of surface-complexed DNA in EPC/DOTAP/DOPE (50/25/25% molar) liposomes and DOTAP/DOPE (50/50% molar) lipoplexes. The study aims to correlate the biological behavior and structural properties of the lipid carriers. We used DNA-hsp65, whose naked action as a gene vaccine against tuberculosis has already been demonstrated. Additionally, surface-complexed DNA-hsp65 in EPC/DOTAP/DOPE (50/25/25% molar) liposomes was effective as a single-dose tuberculosis vaccine. The results obtained showed that the EPC inclusion stabilized the DOTAP/DOPE structure, producing higher melting temperature and lower zeta potential despite a close mean hydrodynamic diameter. Resemblances in morphologies were identified in both structures, although a higher fraction of loaded DNA was not electrostatically bound in EPC/DOTAP/DOPE. EPC also induced a striking reduction in cytotoxicity, similar to naked DNA-hsp65. The proper immune response lead to a polarized antibody production of the IgG2a isotype, even for the cytotoxic DOTAP/DOPE. However, the antibody production was detected at 15 and 30 days for DOTAP/DOPE and EPC/DOTAP/DOPE, respectively. Therefore, the in vivo antibody production neither correlates with the in vitro cytotoxicity, nor with the structural stability alone. The synergistic effect of the structural stability and DNA electrostatic binding upon the surface of structures account for the immunological effects. By adjusting the composition to generate proper packing and cationic lipid/DNA interaction, we allow for the optimization of liposome formulations for required immunization or gene therapy. In a specific manner, our results contribute to studies on the tuberculosis therapy and vaccination732175184FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO – FAPESPsem informaçã

Angiogenesis and lymphangiogenesis in the spectrum of leprosy and its reactional forms.

Angiogenesis and lymphangiogenesis are the processes of neovascularization that evolve from preexisting blood and lymphatic vessels. There are few studies on angiogenesis and none on lymphangiogenesis in leprosy. Thus, the role of neovascularization in the pathophysiological mechanisms of the disease was studied across the spectrum of leprosy, its reactional states and its residual lesions.Seventy-six biopsies of leprosy skin lesions and seven healthy controls were selected. Fifty-five serum samples were used for the detection of CD105 by ELISA. Histological sections were stained with antibodies against CD31 (blood and lymphatic vessels), D2-40/podoplanin (lymphatic vessels), and CD105/endoglin (neovessels). Microvessels were counted in 100 high-power fields (400x) and the number of vessels was evaluated in relation to the extension of the inflammatory infiltrate (0-3), to the bacillary index (0-6) and to the clinical forms. Angiogenesis, as marked by CD31 and CD105, was observed across the leprosy spectrum, compared with the controls. Additionally, there was a positive correlation between these markers with extension of the infiltrate (p <0.0001). For D2/40, lymphangiogenesis was observed in the tuberculoid form (p <0.0001). There was no statistical significance for values of CD105 detected in plasma by ELISA.Angiogenesis is present across the spectrum of leprosy and in its reactional forms. The increase in the number of vessels, as detected by CD31 and CD105 staining, is related to the extension of the inflammatory infiltrate. Samples from reactional lesions have a higher number of CD31+ and CD105+ stained vessels, which indicates their involvement in the pathophysiological mechanisms of the reactional states. The regression of lesions is accompanied by the regression of neovascularization. Drugs inhibiting angiogenesis may be relevant in the treatment of leprosy, in addition to multidrugtherapy, and in the prevention of the development of reactions

Histopathological pictures.

<p>(A) A normal capillary vessel in the papillary dermis, comprising endothelial cells with fusiform shape and showing elongated nuclei (HE, 1000x). (B) The endothelium of an ENL skin lesion, comprising endothelial cells with large and vacuolated cytoplasm, large nuclei, one or more nucleoli and mitoses. The lumen of the capillaries and the interstitium are filled by neutrophils, macrophages, lymphocytes and plasma cells, among other cells (HE-1000x). (C) Capillaries positive for CD31 inside and surrounding the tuberculoid granuloma (IHC, CD31- 200x). (D) Capillaries positive for CD105 inside and surrounding the tuberculoid granuloma (IHC, CD105- 200x). (E) A lepromatous lesion showing multivacuolated histiocytes permeated by capillary vessels (HE- 400x). (F) A residual lesion showing regressive granulomas with a few histiocytes with multivacuolated cytoplasm (HE- 400x). (G) A tuberculoid leprosy case showing a granuloma comprised of epithelioid macrophages in the center and lymphocytes in the periphery (HE- 400x). (H) Lymphatic vessels around the tuberculoid granuloma containing multinucleated giant cells (IHC, D2-40, 200x).</p

Anti-CD105 serum levels detected by ELISA.

<p>Detection of antibodies against CD105 in sera of leprosy patients across the spectrum, in reactional groups and controls. Data reported as means ± SD absorbance. Results show that absorbance was not significantly different between the evaluated groups.</p

Experimental design.

<p>(A) Seventy-six paraffin blocks were selected. The samples constituted punch biopsies of patients in the leprosy spectrum, reactional cases, residual leprosy and controls (n=76). (B) The sections (4µm) were stained by HE and Fite-Faraco (R&J histopathological classification) and (C) immunohistochemistry using antibodies against CD31, D2-40 and CD105 for the counting of microvessels. (D) For each marker, the microvessels were counted in 100 hpf, within the entire thickness of the dermis and part of the subcutaneous tissue. (E) A field immunostained for CD105 in ENL lesion. (*) 46 patients, six TT, six BT, six BB, seven BL, seven LL, seven RR and seven ENL, also had blood samples taken. Nine healthy controls (C) were included in the ELISA test for serum detection of CD105.</p