The use of cell-mediated immunity for the evaluation of influenza vaccines: an upcoming necessity.

Influenza vaccines are a fundamental tool for preventing the disease and reducing its consequences, particularly in specific high-risk groups. In order to be licensed, influenza vaccines have to meet strict criteria established by European Medicines Agency. Although the licensure of influenza vaccines started 65 years ago, Hemagglutination Inhibition and Single Radial Hemolysis are the only serological assays that can ascertain correlates of protection. However, they present evident limitations. The present review focuses on the evaluation of cell-mediated immunity (CMI), which plays an important role in the host immune response in protecting against virus-related illness and in the establishment of long-term immunological memory. Although correlates of protection are not currently available for CMI, it would be advisable to investigate this kind of immunological response for the evaluation of next-generation vaccines.

Increase of chromium tolerance in Scenedesmus acutus after sulfur starvation: Chromium uptake and compartmentalization in two strains with different sensitivities to Cr(VI).

In photosynthetic organisms sulfate constitutes the main sulfur source for the biosynthesis of GSH and its precursor Cys. Hence, sulfur availability can modulate the capacity to cope with environmental stresses, a phenomenon known as SIR/SED (Sulfur Induced Resistance or Sulfur Enhanced Defence). Since chromate may compete for sulfate transport into the cells, in this study chromium accumulation and tolerance were investigated in relation to sulfur availability in two strains of the unicellular green alga Scenedesmus acutus with different Cr-sensitivities. Paradoxically, sulfur deprivation has been demonstrated to induce a transient increase of Cr-tolerance in both strains. Sulfur deprivation is known to enhance the sulfate uptake/assimilation pathway leading to important consequences on Cr-tolerance: (i) reduced chromate uptake due to the induction of high affinity sulfate transporters (ii) higher production of cysteine and GSH which can play a role both through the formation of unsoluble complexes and their sequestration in inert compartments. To investigate the role of the above mentioned mechanisms, Cr accumulation in total cells and in different cell compartments (cell wall, membranes, soluble and miscellaneous fractions) was analyzed in both sulfur-starved and unstarved cells. Both strains mainly accumulated chromium in the soluble fraction, but the uptake was higher in the wild-type. In this type a short period of sulfur starvation before Cr(VI) treatment lowered chromium accumulation to the level observed in the unstarved Cr-tolerant strain, in which Cr uptake seems instead less influenced by S-starvation, since no significant decrease was observed. The increase in Cr-tolerance following S-starvation seems thus to rely on different mechanisms in the two strains, suggesting the induction of a mechanism constitutively active in the Cr-tolerant strain, maybe a high affinity sulfate transporter also in the wild-type. Changes observed in the cell wall and membrane fractions suggest a strong involvement of these compartments in Cr-tolerance increase following S-starvation.

Sea buckthorn bud extract displays activity against cell-cultured Influenza virus.

INTRODUCTION: Vaccines and antiviral drugs are the most widely used methods of preventing or treating Influenza virus infection. The role of sea buckthorn (SBT) bud dry extract as a natural antiviral drug against Influenza was investigated. METHODS: Influenza virus was cultured in the MDCK cell line, with or without SBT bud extract, and virus growth was assessed by HA and TCID50 virus titration in terms of cytopathic effect on cells. Several concentrations of extract were tested, the virus titer being measured on day 4 after infection. RESULTS: After infection, the virus titer in the control sample was calculated to be 2.5 TCID50/ml; treatment with SBT bud extract reduced the virus titer to 2.0 TCID50/ml at 50 µg/ml, while the HA titer was reduced from 1431 (control) to 178. Concentrations lower than 50µg/ml displayed an inhibitory effect in the HA assay, but not in the TCID50 virus titration; however, observation of the viral cultures confirmed a slowdown of viral growth at all concentrations. DISCUSSION: Natural dietary supplements and phytotherapy are a growing market and offer new opportunities for the treatment of several diseases and disorders. These preliminary experiments are the first to show that SBT bud extract is able to reduce the growth of the Influenza A H1N1 virus in vitro at a concentration of 50 µg/ml. This discovery opens up the possibility of using SBT bud extract as a valid weapon against Influenza and, in addition, as the starting-point for the discovery of new drugs.

Cytokinin-like activity of N,N'-diphenylureas. N,N'-bis-(2,3-methylenedioxyphenyl)urea and N,N'-bis-(3,4-methylenedioxyphenyl)urea enhance adventitious root formation in apple rootstock M26 (Malus pumila Mill.).

Vegetative propagation of cuttings is a widespread method to multiplicate plants. Adventitious root formation is a key step in vegetative propagation and considerable progress has recently been made in understanding root formation. But, in spite of the efforts made, no new rooting treatments have been developed. Here, we report for the first time, that N,N'-bis-(2,3-methylenedioxyphenyl)urea and N,N'-bis-(3,4-methylenedioxyphenyl)urea enhance adventitious root formation in microcuttings of Malus pumila Mill. rootstock M26. Roots emerge without auxin supplementation in the darkness, transfer in hormone free medium, or callus formation. With the use of different bioassays, we also demonstrate that these two diphenylurea derivatives do not show cytokinin- or auxin-like activity.

New potential markers of in vitro tomato morphogenesis identified by mRNA differential display.

The identification of plant genes involved in early phases of in vitro morphogenesis can not only contribute to our understanding of the processes underlying growth regulator-controlled determination, but also provide novel markers for evaluating the outcome of in vitro regeneration experiments. To search for such genes and to monitor changes in gene expression accompanying in vitro regeneration, we have adapted the mRNA differential display technique to the comparative analysis of a model system of tomato cotyledons that can be driven selectively toward either shoot or callus formation by means of previously determined growth regulator supplementations. Hormone-independent transcriptional modulation (mainly down-regulation) has been found to be the most common event, indicating that a non-specific reprogramming of gene expression quantitatively predominates during the early phases of in vitro culture. However, cDNA fragments representative of genes that are either down-regulated or induced in a programme-specific manner could also be identified, and two of them (G35, G36) were further characterized. One of these cDNA fragments, G35, corresponds to an mRNA that is down-regulated much earlier in callus- (day 2) than in shoot-determined explants (day 6). The other, G36, identifies an mRNA that is transiently expressed in shoot-determined explants only, well before any macroscopic signs of differentiation become apparent, and thus exhibits typical features of a morphogenetic marker.

Comparison between a normal and a Cr-tolerant strain of Scenedesmus acutus as a food source to Daphnia magna.

A normal (S2-N) and a Cr-tolerant (S2-T) strain of Scenedesmus acutus, grown in Cr-free medium or in the presence of 1, 5, or 10 mg/liter Cr(VI), were fed to Daphnia magna. An increase of growth and fecundity was observed in the daphnids when the algae had undergone an increase of dry mass following Cr treatment, which happened in S2-N in the presence of 1 mg/liter and in S2-T in the presence of 5 mg/liter. When the algae were pretreated with 10 mg/liter, growth and fecundity were reduced in the daphnids fed on S2-N, but remained normal in the daphnids fed on S2-T. It is concluded that the tolerant strain remains a good food source even if grown in the presence of a Cr concentration that reduces the nutritional value of the normal strain.

Chromium-induced sexual reproduction gives rise to a Cr-tolerant progeny in Scenedesmus acutus.

A clonal population of Scenedesmus acutus was treated for 3 months with 1 mg/liter Cr(VI) and then returned to Cr-free medium. After several months, the cells were treated with different Cr concentrations and then subjected to a series of morphological observations and metabolic tests. The results, compared with those obtained with a normal cell population treated in the same way, demonstrate that the progeny of algae subjected to the prolonged Cr treatment has acquired tolerance to the metal, as it can survive and grow in the presence of Cr concentrations that are lethal to normal cells. An ultrastructural description of gametes and zygotes is also given.

ADVANCE and ADAM: two algorithms for the analysis of global similarity between homologous informational sequences.

Two algorithms for the analysis of global similarity between sequences of informational polymeric molecules (nucleic acids and proteins) are proposed: one (ADVANCE) merely gives a quantification of the global similarity between two sequences, and is very fast; the other (ADAM) also provides an alignment of the sequences. Both are new algorithms, implement Sellers' theorem, do not require parameters, are able to analyze two sequences of 32,000 elements each, and are fast; nevertheless, they differ deeply in the algorithm, in the programming language type, and in their planning, and will thus have to be treated separately. In fact, the different nature of the required output in each program causes the common aim (to obtain power and speed) to be reached only through very different ways and methods.

Effect of 1,2-benzisoxazole-3-acetic acid on plant regeneration from protoplasts of Nicotiana tabacum.

Benzisoxazole-3-acetic acid, a new synthetic growth regulator, was administered to protoplast cultures from Nicotiana tabacum and subsequently to the developed microcalluses, to test its activity on plant regeneration from protoplasts in different culture conditions. Such activity, compared to that of naphthalene-acetic acid, proved to be rather low in the stage of cellular division and microcallus formation but particulary high in the stage of shoot induction from microcallus, thus confirming that the activity of this compound is mainly morphogenetic.

Non-radioactive detection of ß-glucuronidase and chloramphenicol acetyltransferase activities in co-transformed protoplasts by HPLC.

The use of transient gene expression assays for the study of natural or engineered plant promoters is affected by a considerable degree of inter-experiment variability. As a means of obtaining interpretable data from a limited number of experiments, we worked out conditions for the simultaneous determi nation of the activity of two reporter genes, a "sample" and a "reference", ona single extract of co-transformed protoplasts. ß-glucuronidase (GUS) and chloramphenicol acetyl transferase (CAT) genes, both under the control of the CaMV 35S promoter, were transferred into tobacco (Nicotiana tabacum L.) protoplasts on two independent plasmids. The parallel expression of the two reporter genes in several independent co-transformation experiments was verified. Conditions for the use of a single protoplast extraction buffer and for the simultaneous assay of both reporter gene activities were set up. A HPLC method for the non-radioactive determination of both enzyme activities on a single aliquot of the reaction mixture was developed. The resulting procedure was tested using the GUS gene as "reference" and the CAT gene, under the control of either wild type or upstream-deleted (-90) CaMV 35S promoter, as "sample". The protocol is simple and allows the fast analysis of plant promoters in the presence of a true internal standard under conditions in which assay manipulations are reduced to a minimum and both reporter gene activities are subjected to the same experimental treatments.

A four-state cross bridge model for muscle contraction. Mathematical study and validation.

A mathematical model of contracting skeletal muscle is studied. The model is composed by an elastic element (SE) in series with a contractile element (CE) that describes the cross bridge kinetics with a formulation derived by that proposed by Eisenberg and Hill (1978). An analytical study of the system of nonlinear partial differential equations of the model allows the existence and the uniqueness of the solution to be proved. A suitable approach to the numerical solution is defined and a series of numerical tests are performed. These tests lead to select an appropriate set of parameters and allow to compare model predictions and experimental observations on frog skeletal muscle.