IL-12 and GM-CSF in DNA/MVA immunizations against HIV-1 CRF12_BF Nef induced T-cell responses with an enhanced magnitude, breadth and quality

In Argentina, the HIV epidemic is characterized by the co-circulation of subtype B and BF recombinant viral variants. Nef is an HIV protein highly variable among subtypes, making it a good tool to study the impact of HIV variability in the vaccine design setting. We have previously reported a specific cellular response against NefBF with low cross-reactivity to NefB in mice. The aim of this work was to analyze whether the co-administration of IL-12 and GM-CSF, using DNA and MVA vaccine vectors, could improve the final cellular response induced. Mice received three DNA priming doses of a plasmid that express NefBF plus DNAs expressing IL-12 and/or GM-CSF. Afterwards, all the groups were boosted with a MVAnefBF dose. The highest increase in the magnitude of the NefBF response, compared to that induced in the control was found in the IL-12 group. Importantly, a response with higher breadth was detected in groups which received IL-12 or GM-CSF, evidenced as an increased frequency of recognition of homologous (BF) and heterologous (B) Nef peptides, as well as a higher number of other Nef peptide pools representing different viral subtypes. However, these improvements were lost when both DNA cytokines were simultaneously administered, as the response was focused against the immunodominant peptide with a detrimental response towards subdominant epitopes. The pattern of cytokines secreted and the specific-T-cell proliferative capacity were improved in IL-12 and IL-12+GM-CSF groups. Importantly IL-12 generated a significant higher T-cell avidity against a B heterologous peptide.This study indicates that the incorporation of DNA expressing IL-12 in DNA/MVA schemes produced the best results in terms of improvements of T-cell-response key properties such as breadth, cross-reactivity and quality (avidity and pattern of cytokines secreted). These relevant results contribute to the design of strategies aimed to induce T-cell responses against HIV antigens with higher quality.     

X-ray and neutron small-angle scattering analysis of the complex formed by the Met receptor and the Listeria monocytogenes invasion protein InlB

The Listeria monocytogenes surface protein InlB binds to the extracellular domain of the human receptor tyrosine kinase Met, the product of the c-met proto-oncogene. InlB binding activates the Met receptor, leading to uptake of Listeria into normally nonphagocytic host cells. The N-terminal half of InlB (InlB₃₂₁) is sufficient for Met binding and activation. The complex between this Met-binding domain of InlB and various constructs of the Met ectodomain was characterized by size exclusion chromatography and dynamic light scattering, and structural models were built using small-angle X-ray scattering and small-angle neutron scattering. Although most receptor tyrosine kinase ligands induce receptor dimerization, InlB₃₂₁ consistently binds the Met ectodomain with a 1:1 stoichiometry. A construct comprising the Sema and PSI domains of Met, although sufficient to bind the physiological Met ligand hepatocyte growth factor/scatter factor, does not form a complex with InlB₃₂₁ in solution, highlighting the importance of Met Ig domains for InlB binding. Small-angle X-ray scattering and small-angle neutron scattering measurements of ligand and receptor, both free and in complex, reveal an elongated shape for the receptor. The four Ig domains form a bent, rather than a fully extended, conformation, and InlB₃₂₁ binds to Sema and the first Ig domain of Met, in agreement with the recent crystal structure of a smaller Met fragment in complex with InlB₃₂₁. These results call into question whether receptor dimerization is the basic underlying event in InlB₃₂₁-mediated Met activation and demonstrate differences in the mechanisms by which the physiological ligand hepatocyte growth factor/scatter factor and InlB₃₂₁ bind and activate the Met receptor.     

Rapid MALDI-TOF-MS analysis in the study of interaction between whole bacterial cells and human target molecules: binding of Bifidobacterium to human plasminogen

MALDI-TOF (Matrix Assisted Laser Desorption Ionization-Time of Flight)—mass spectrometry has been applied, for the first time, in the investigation of whole Bifidobacterium cells-host target proteins interaction. In particular, by means of this technique, a dose dependent human plasminogen-binding activity has been shown for Bifidobacterium. The involvement of lysine binding sites on the bacterial cell surface has been proved. The obtained result was found to be consistent with that from well-established standard methodologies, thus the proposed MALDI-TOF approach has the potential to enter as a fast alternative method in the field of biorecognition studies involving in bacterial cells and proteins of human origin.     

Enhanced affinity of ketotifen toward tamarind seed polysaccharide in comparison with hydroxyethylcellulose and hyaluronic acid: a nuclear magnetic resonance investigation

Nuclear magnetic resonance (NMR) spectroscopy demonstrated that, in aqueous solution, ketotifen fumarate bound more strongly to tamarind seed polysaccharide (TSP) than to hydroxyethylcellulose or hyaluronic acid. Results were confirmed by dynamic dialysis technique.     

A combinatorial biocatalysis approach to an array of cholic acid derivatives

A 39-member library of bile acid derivatives was prepared starting from 3alpha,7alpha,12alpha-trihydroxy-5beta-cholan-24-oic acid methyl ester using a combinatorial biocatalytic approach. A regioselective oxidation step, catalyzed by hydroxysteroid dehydrogenases, followed by an acylation step with a series of different acyl donors catalyzed by Candida antarctica lipase B, led to the modification of the bile acid scaffold. Each member of the library was obtained in high purity and good yield.     

The effect of manganese supply on exudation of carboxylates by roots of lucerne (Medicago sativa)

Some low-molecular-weight carboxylates commonly found in plant root exudates have the potential to increase the availability of Mn in the rhizosphere. Release of various compounds into the rhizosphere by plant roots may also be a mechanism by which certain species and genotypes are able to tolerate conditions of low Mn availability better than others. Lucerne (Medicago sativa L.) plants of Salado, a genotype tolerant to Mn deficiency, and Sirosal, an intolerant genotype, were grown in solution culture with 0, 5 or 500 nM Mn (Mn-0, Mn-5 and Mn-500). Exudates of whole root systems were collected at 14, 24 and 36 d and analysed by HPLC. Oxalate, tartarate, L-malate, lactate, malonate, maleate, citrate and succinate were detected and quantified in exudates under all Mn treatments. Malonate, citrate and succinate accounted for the majority of carboxylates in the exudates. Exudation increased with plant age, but amounts of individual carboxylates remained constant in proportion to the total amount exuded. A significant increase in exudation of all carboxylates other than malonate and maleate resulted from omission of Mn from nutrient solutions. Salado exuded more oxalate, tartarate, L-malate, lactate, citrate and succinate than Sirosal at Mn-0, and more citrate and succinate than Sirosal at Mn-5. Genotypic differences in carboxylate exudation under Mn-0 were associated with production of roots with diameter <100 μm. Plant Mn concentrations and growth rates suggested carboxylate exudation differences were not the sole factor responsible for differential tolerance to Mn deficiency in the lucerne genotypes.     

Discovery of the first small molecule inhibitor of human DDX3 specifically designed to target the RNA binding site: towards the next generation HIV-1 inhibitors

Efficacy of currently approved anti-HIV drugs is hampered by mutations of the viral enzymes, leading invariably to drug resistance and chemotherapy failure. Recent data suggest that cellular co-factors also represent useful targets for anti-HIV therapy. Here we describe the identification of the first small molecules specifically designed to inhibit the HIV-1 replication by targeting the RNA binding site of the human DEAD-Box RNA helicase DDX3. Optimization of a easily synthetically accessible hit (1) identified by application of a high-throughput docking approach afforded the promising compounds 6 and 8 which proved to inhibit both the helicase and ATPase activity of DDX3 and to reduce the viral load of peripheral blood mononuclear cells (PBMC) infected with HIV-1.     

HCC Development Is Associated to Peripheral Insulin Resistance in a Mouse Model of NASH

NAFLD is the most common liver disease worldwide but it is the potential evolution to NASH and eventually to hepatocellular carcinoma (HCC), even in the absence of cirrhosis, that makes NAFLD of such clinical importance. Aim: we aimed to create a mouse model reproducing the pathological spectrum of NAFLD and to investigate the role of possible co-factors in promoting HCC. Methods: mice were treated with a choline-deficient L-amino-acid-defined-diet (CDAA) or its control (CSAA diet) and subjected to a low-dose i.p. injection of CCl₄ or vehicle. Insulin resistance was measured by the euglycemic-hyperinsulinemic clamp method. Steatosis, fibrosis and HCC were evaluated by histological and molecular analysis. Results: CDAA-treated mice showed peripheral insulin resistance at 1 month. At 1–3 months, extensive steatosis and fibrosis were observed in CDAA and CDAA+CCl₄ groups. At 6 months, equal increase in steatosis and fibrosis was observed between the two groups, together with the appearance of tumor. At 9 months of treatment, the 100% of CDAA+CCl₄ treated mice revealed tumor versus 40% of CDAA mice. Insulin-like Growth Factor-2 (IGF-2) and Osteopontin (SPP-1) were increased in CDAA mice versus CSAA. Furthermore, Immunostaining for p-AKT, p-c-Myc and Glypican-3 revealed increased positivity in the tumors. Conclusions: the CDAA model promotes the development of HCC from NAFLD-NASH in the presence of insulin resistance but in the absence of cirrhosis. Since this condition is increasingly recognized in humans, our study provides a model that may help understanding mechanisms of carcinogenesis in NAFLD.     

Rat monoclonal antibodies to rabbit and human serum low-density lipoprotein.

A total of 16 hybrid myeloma clones secreting monoclonal antibodies (McAb) to rabbit or human serum low-density lipoprotein (LDL) were derived from the fusion of spleen cells from LOU or DA rats immunized with rabbit or human LDL and the rat myeloma lines Y3 Ag1.2.3 or YB2/0. Anti-(rabbit LDL) McAb showed limited reactivity with LDL from human, rhesus-monkey, rat and mouse serum. Six out of seven anti-(human LDL) McAb reacted with rhesus-monkey LDL, and only one showed partial cross-reaction with rabbit LDL. Binding-competition experiments indicated that the epitopes recognized by the anti-(rabbit LDL) IgG could be grouped into two major clusters: McAb in the first cluster reacted either with apo-(lipoprotein B-100) (apoB-100) and apo-(lipoprotein B-74) (apoB-74) or with apoB-100 but not with apo-(lipoprotein B-48) (apoB-48), the lower-Mr form of apoB of intestinal origin; the McAb in the second cluster all reacted with apoB-48 in addition to apoB-100 or apoB-100 and apoB-74. The six anti-(human LDL) IgG bound to separate epitopes on LDL. Further data on the epitope specificity of these McAb were obtained by antibody blotting after partial proteolysis of apoB-100 with trypsin or staphylococcal V8 proteinase, and the data confirmed the results obtained with the binding-competition experiments. One McAb to rabbit LDL inhibited the binding of LDL to the fibroblast LDL receptor (50% inhibition at a McAb/LDL molar ratio of 10). A similar result was produced by two other McAb at higher concentrations of antibody.     

Deep placement of manganese fertiliser improves sustainability of lucerne growing on bauxite residue: A glasshouse study

Successful revegetation of bauxite residue sand (BRS) requires large inputs of nutrients such as manganese (Mn), yet Mn deficiency is still encountered, raising doubts about sustainable revegetation of BRS disposal areas. The application of deep placement of Mn, a measure common in agriculture, was examined as a method for improving productivity and sustainability when lucerne (Medicago sativa L.) is used as a species for BRS revegetation. In pots containing BRS, Mn was banded at 2.5-, 10- and 20-cm depths at rates of 10, 20 and 50 g g^–1 BRS. Two lucerne genotypes used were Salado, a Mn-deficiency-tolerant variety, and Sirosal, a Mn-deficiency-sensitive variety. Banding at 10-cm depth produced the best shoot growth of Sirosal at each Mn rate. Greatest shoot growth in Salado was found at 2.5-, 10- and 20-cm depths for 10, 20 and 50 g Mn g^–1 BRS, respectively. Deep banding 20 g Mn g^–1 BRS at 10-cm depth significantly increased lucerne growth compared with mixing through the profile. Banding at 20 cm produced Mn deficiency symptoms in lucerne during early growth, but symptoms were alleviated when sufficient amounts of roots proliferated in the banding zone. Dissolution and movement of Mn away from the fertiliser band were also investigated. In pots without plants, water throughput from watering twice weekly to 110% field capacity had no effect on the amount of extractable Mn at distances more than 1 cm away from the original Mn band position. Whilst not only providing a more effective supply of Mn for BRS revegetation over one growth period, deep-banding of adequate rates of Mn may also result in a longer residual value, reducing the need for frequent broadcast applications.