Alpha 1-antitrypsin variants produced by recombinant DNA: differences in elastase inhibitory activity and resistance to oxidant agents

Inherited or "acquired" deficiency of alpha 1-antitrypsin (believed to be the cause of pulmonary emphysema) will probably be treated in the future by replacement with alpha 1-antitrypsin purified from human plasma or produced by recombinant DNA, which seems promising because it permits site-specific mutagenesis in the oxidizable active site of the normal human alpha 1-antitrypsin. The aim of this in-vitro study was to investigate the elastase inhibitory activity and the resistance to oxidizing agents of normal human alpha 1-antitrypsin, a recombinant yeast-produced variant (VAL 358) and a recombinant E. coli-produced variant (LEU 358). The inhibitors were exposed to chemical oxidants (NCS, H2O2, xanthine/xanthine oxidase, chloramine-T) and to PMA-activated neutrophils. The elastase inhibitory activity was assayed on porcine pancreatic elastase and neutrophil elastase. Normal alpha 1-antitrypsin and VAL 358 variant were good inhibitors of both elastases. LEU 358 variant was the best inhibitor for neutrophil elastase, but it poorly inhibited the porcine pancreatic elastase. Normal alpha 1-antitrypsin was affected by all oxidants; both variants were almost totally resistant to chemical oxidants and to activated neutrophils. We conclude that recombinant alpha 1-antitrypsin variants differ in their elastase inhibitory activity and offer increased resistance to oxidant agents.     

L’utilizzo della fertomcidina U in alcuni casi di patologia della mucosa orale

Objectives

In this work the authors have studied the use of Fertomcidina U in the treatment of angular cheilitis and hard palate candidosis.

Results and conclusions

This pharmacological approach has been really useful both as antalgic action in the immediate treatment and anti-inflammatory in the long term results.     

Sustained virologic response to direct-acting antiviral agents predicts better outcomes in hepatitis C virus-infected patients: A retrospective study

BACKGROUND Direct-acting antiviral agents(DAAs) are extremely effective in eradicating hepatitis C virus(HCV) in chronically infected patients. However, the protective role of the sustained virologic response(SVR) achieved by second-and thirdgeneration DAAs against the onset of hepatocellular carcinoma(HCC) and mortality is less well established.AIM To examine the occurrence of HCC or death from any cause in a retrospectiveprospective study of patients treated with DAAs.METHODS Patients were enrolled from a tertiary academic hospital center for liver disease management that collects subject data mainly from northeastern Italy. The study was conducted in 380 patients(age: 60 ± 13 years, 224 males, 32% with cirrhosis)treated with DAAs with or without SVR(95/5%), with a median follow up of 58 wk(interquartile range: 38-117). The baseline anthropometric features, HCV viral load, severity of liver disease, presence of extra-hepatic complications, coinfection with HIV and/or HBV, alcohol consumption, previous interferon use, alphafetoprotein levels, and renal function were considered to be confounders.RESULTS The incidence rate of HCC in patients with and without SVR was 1.3 and 59 per100 person-years, respectively(incidence rate ratio: 44, 95%CI: 15-136, P 0.001).Considering the combined endpoint of HCC or death from any cause, the hazard ratio(HR) for the SVR patients was 0.070(95%CI: 0.025-0.194, P 0.001). Other independent predictors of HCC or death were low HCV viremia(HR: 0.808, P =0.030), low platelet count(HR: 0.910, P = 0.041), and presence of mixed cryoglobulinemia(HR: 3.460, P = 0.044). Considering SVR in a multi-state model,the independent predictors of SVR achievement were absence of cirrhosis(HR:0.521, P 0.001) and high platelet count(HR: 1.019, P = 0.026). Mixed cryoglobulinemia predicted the combined endpoint in patients with and without SVR(HR: 5.982, P = 0.028 and HR: 5.633, P = 0.047, respectively).CONCLUSION DAA treatment is effective in inducing SVR and protecting against HCC or death.A residual risk of HCC persists in patients with advanced liver disease or with complications, such as mixed cryoglobulinemia or renal failure.     

Intrinsic and microenvironmental defects are involved in the age-related changes of Lin - c-kit+ hematopoietic progenitor cells

The aim of this study was to evaluate through cross-transplantation models the effect of aging on the number of Lin(-)c-kit+ hematopoietic progenitor cells, on their ability to differentiate towards a lymphocyte phenotype, and on the role of the microenvironment in hematopoietic differentiation. The absolute number of purified Lin(-)c-kit+ cells from bone marrow was significantly lower in aged than in young mice. When transplanted in young recipients, Lin(-)c-kit+ hematopoietic progenitor cells from aged mice showed a reduced differentiation capacity in T cells and NK cells, compared to Lin(-)c-kit+ cells from young animals. The role of microenvironment in Lin(-)c-kit+ hematopoietic progenitor cells differentiation was evaluated by injecting young Lin(-)c-kit+ cells in young or aged recipients, the latter transplanted or not with a young thymus. In these conditions, the differentiation of Lin(-)c-kit+ cells from young mice in T and NK cells was less efficient in aged than in young recipients, independently of thymus grafting in aged recipients. In addition to quantitative defects qualitative alterations were also present in Lin(-)c-kit+ cells from aged mice, as evidenced by the fact that the injection of Lin(-)c-kit+ cells from aged donors in young recipients differentiated in CD4+ T cells that retained an interleukin-4 (IL-4) production in-between young and old control values. In conclusion, we have demonstrated that aging is associated with numerical and functional alterations of Lin(-)c-kit+ hematopoietic progenitor cells as well as with an altered microenvironment that is required for Lin(-)c-kit+ cells differentiation toward a lymphocyte phenotype.     

Imiquimod and S-27609 as adjuvants of DNA vaccination in a transgenic murine model of HER2/neu-positive mammary carcinoma

DNA vaccination against HER-2/neu is an effective way to induce an immune response able to oppose the spontaneous development of mammary tumours occurring in HER-2/neu transgenic mice. In this study, we have evaluated the potential of Imiquimod and the analogue S-27609 as adjuvants of DNA vaccination against HER-2/neu in transgenic mice. The association of a DNA vaccine encoding a portion of rat HER2/neu with either Imiquimod or S-27609 was found to delay the development of spontaneous mammary tumours and to reduce their incidence, in comparison with DNA vaccination alone. Almost 80 or 40% of tumour-free mice were found at the end of measurement time in mice vaccinated and supplemented with Imiquimod or S-27609, respectively. The antitumour preventive effect was associated with increased antibody and cell-mediated immune responsiveness against HER-2/neu. In mice vaccinated and supplemented with Imiquimod, a small but significant increase of rat p185neu-specific cytotoxicity and of IFN-gamma and IL-2-producing CD8T cells, together with a reduction of IL-4-producing CD4T cells, and a switch from an IgG1 towards a IgG2a phenotype of anti-p185neu antibodies, suggested a TH1 polarization of the immune response. The immunoregulatory efficacy of S-27609 was lower than that observed for Imiquimod. These data highlight the potential of Imiquimod, and, to a lower extent, of S-27609, as immunological adjuvants of therapeutic DNA vaccines.     

Phenotype,antigen-presenting capacity,and migration of antigen-presenting cells in young and old age

In the present paper, we investigated whether the phenotype, the antigen-presenting capacity, and the migration of antigen-presenting cells (APCs), are affected by the aging process. APCs were obtained incubating peritoneal monocyte-macrophage cells with granulocyte-macrophage colony-stimulating factor (GM-CSF) (immature APCs) or GM-CSF and IFNgamma (mature APCs). Phenotypically, after 8 days incubation, APCs cultures were composed of CD11c and Mac-3 cells, with a similar representation, both in young and old mice. The absolute number and the expression of MHC I and II, CD80, and CD86 both on immature and mature APCs were not significantly different in young and old mice. APCs from old mice induced similar lymphocyte proliferative responses but lower lymphocyte cytotoxicity and a reduced number of CD8(+) T cells producing IFNgamma in comparison with APCs from young animals. Lymphocyte responses were antigen-specific, since TS/A pulsed APCs induced lymphocyte cytotoxicity against TS/A but not against syngeneic TUBO tumor cells. The low expression of the mRNA for the migratory CCR7 chemokine receptor present in immature APCs from old mice was greatly increased in mature APCs up to the levels found in APCs from young animals. The in vivo migration of APCs was higher in old than in young mice. These results demonstrate that some alterations in APCs function are present in aging, suggesting that an increased migratory capacity of old APCs may be required to balance their reduced antigen presentation to cytotoxic lymphocytes.     

Linomide blocks angiogenesis by breast carcinoma vascular endothelial growth factor transfectants.

The blocking of angiogenesis provides a novel therapeutic target to inhibit tumour spreading. In this study, we investigated the effect of linomide on angiogenesis induced in vivo by highly angiogenic breast carcinoma cells. The rabbit cornea was used to assess neovascular growth in the absence of a tumour mass. MCF-7 cells stably transfected with the cDNA encoding for vascular endothelial growth factor 121 (VEGF121) (V12 clone) were used to elicit a potent VEGF-dependent corneal angiogenesis. After tumour cell implant, albino rabbits received 100 mg kg(-1) day(-1) linomide for 5 consecutive days. Daily observation of neovascular progression indicated that linomide blocked angiogenesis. The antiangiogenic effect of linomide was apparent within 48 h from the beginning of the treatment and was both angiosuppressive and angiostatic. The block of neovascular growth lasted over 10 days from treatment suspension, and preformed vessels, which had regressed, remained dormant, suggesting the persistence of unfavourable conditions for capillary progression. Linomide (50-200 microg ml[-1]) was not cytotoxic in vitro on resting capillary endothelial cells but blocked endothelial cell replication induced by VEGF. Our data indicate that linomide can efficiently and persistently block VEGF-dependent angiogenesis in vivo in the absence of a growing tumour mass. These data suggest that linomide could be a chemopreventive drug in breast cancer patients and a valuable tool in clinical settings in which metastatic spreading occurs in the absence of a detectable tumour mass.     

The mechanisms of apoptosis in biology and medicine: a new focus for ophthalmology

Defects in apoptosis (programmed cell death) have recently emerged as being closely involved in the pathogenesis of most ocular diseases and, therefore, apoptosis is now a topic of exponential interest in ophthalmology. This review summarizes recent works on mechanisms of apoptosis, from its initiation and modulation to the switching-on of its execution machinery. Interactions of cell death with cell division programs to orchestrate ontogenesis, aging, and adult life and their alterations in human diseases are pointed out. Two main apoptotic signaling pathways are identified: a death receptor-dependent (extrinsic) pathway and a mitochondrion-dependent (intrinsic) pathway. Mitochondrion harbors both antiapoptotic (Bcl-2, Bcl-XL) and apoptotic factors (Smac/Diablo, Apaf-1, cytochrome c). Its permeability transition pore (mPTP) is the main trigger of cell suicide. The process of mPTP opening, in association with extrusion to cytoplasm of a variety of apoptotic factors, is shown. Cytochrome c is one of these apoptotic factors. When expelled to cytoplasm, this double-faced respiratory chain component assembles with two other modules, Apaf-1 and procaspase 9, to form a protein complex--the apoptosome--that starts apoptosis execution. Another respiratory chain component, the CoQ10, is believed to counteract mPTP opening. What makes apoptosis particularly exciting for medicine is that its dysfunctions play a central role in the pathogenesis of several human diseases. For instance, excesses of apoptosis lead to cell loss that accompanies neurodegenerative diseases, whereas genetically determined defects of apoptosis lead to the deregulated cell proliferation typical of cancer. A variety of ophthalmologic diseases, such as post-keratectomy haze, corneal lesions, cataract, glaucoma, senile maculopathies, and genetic ocular pathologies, that underlie apoptosis dysfunctions are treated in detail in the other reviews of this issue.     

Effect of resveratrol on the development of spontaneous mammary tumors in HER-2/neu transgenic mice

Resveratrol (Res) has been reported to possess cancer chemopreventive activity on the basis of its in vitro effects on tumor cells and in vivo experimental models of rodents transplanted with parental tumors or treated with carcinogens. We investigated the effects of Res on the development of mammary tumors appearing spontaneously in HER-2/neu transgenic mice at an early age. The mechanisms involved in the Res antitumor effect were evaluated by studying the immune effectiveness, tumor apoptosis and expression of mRNA and protein for HER-2/neu in tumoral mammary glands from Res-treated mice and in tumor cell lines. Res supplementation delayed the development of spontaneous mammary tumors (p < 0.001), reduced the mean number and size of mammary tumors (p < 0.0001) and diminished the number of lung metastases in HER-2/neu transgenic mice. The effects of Res were associated with downregulation of HER-2/neu gene expression and increased apoptosis both in tumoral mammary glands and in murine (N202) and human (SKBr3) tumor cell lines. Neither the basal, the IL-2-induced NK activity nor the lymphocyte number and proliferation was modified in Res-supplemented compared to control mice. Our results demonstrate that Res supplementation delays the development and reduces the metastasizing capacity of spontaneous mammary tumors in HER-2/neu transgenic mice. The antitumor effect of Res might be related to the downregulation of HER-2/neu expression and the induction of apoptosis in tumor cells.