PD-1 /PD-L1 checkpoint in hematological malignancies.

Programmed cell death protein 1 (PD-1), is a cell surface receptor with an important role in down-regulating the immune system and promoting self-tolerance by suppressing T cell inflammatory activity. PD-1/PDL1 axis represents a checkpoint to control immune responses and it is often used as a mechanism of immune escaping by cancers and infectious diseases. Many data demonstrate its important role in solid tumors and report emerging evidences in lymphoproliferative disorders. In this review, we summarized the available data on the role of PD-1/PD-L1 checkpoint in lymphoproliferative diseases and the therapeutics use of monoclonal blocking antibodies.

Phylogeny of the genus Azospirillum based on 16S rDNA sequence.

The 16S rDNA of 17 strains of Azospirillum, 14 assigned to one of the known species A. amazonense, A. brasilense, A. halopraeferens, A. irakense and A. lipoferum, and the other three of uncertain taxonomic position, was sequenced after polymerase chain reaction amplification and analysed in order to investigate the phylogenetic relationships at the intra-generic and super-generic level. The phylogenetic analysis confirms that the genus Azospirillum constitutes a phylogenetically separate entity within the alpha subclass of Proteobacteria and that the five species are well defined. A. brasilense and A. lipoferum are closely related species and form one cluster together with A. halopraeferens; the pair of species A. amazonense and A. irakense forms a second cluster in which Rhodospirillum centenum is also placed.

Identification of Azospirillum strains by restriction fragment length polymorphism of the 16S rDNA and of the histidine operon.

DNA fingerprints of several Azospirillum strains, belonging to the five known species A. amazonense, A. brasilense, A. halopraeferens, A. irakense and A. lipoferum, were obtained by restriction analysis of the amplified 16S rDNA and by restriction fragment length polymorphism of the histidine biosynthetic genes. Data obtained showed that amplified rDNA restriction analysis is an easy, fast, reproducible and reliable tool for identification of Azospirillum strains, mainly at the species level, whereas restriction fragment length polymorphism could, in some cases, differentiate strains belonging to the same species. Moreover, both analyses gave congruent results in grouping strains and in the assignment of new strains to a given species.

Control of nifH transcription in Azospirillum brasilense: involvement of NifA and of cis-acting sequences.

The regulatory sequences of Azospirillum brasilense Sp7 nifH gene were fused with the cam reporter gene and used for studying the factors controlling nifH transcription. A DNA sequence, downstream the ATG codon of nifH, that could be involved in the negative regulation of nifH transcription, was identified. The effect of 1 and 2 mM of ammonium on the transcription of the A. brasilense nifH gene and on the nitrogenase activity, in the presence of the Klebsiella pneumoniae NifA protein, was examined.

Use of random amplified polymorphic DNA (RAPD) for generating specific DNA probes for microorganisms.

We report the rapid generation of DNA probes for several Azospirillum strains. This method does not require any knowledge of the genetics and/or the molecular biology of the organism (genome) to be investigated. The procedure is based on the generation of random amplified polymorphic DNA (RAPD) fingerprints using primers with an embedded restriction site. The amplification product(s) peculiar to one strain or common to two or more strains can be purified, cloned, sequenced and used as molecular probes in hybridization experiments for the detection and identification of microorganisms. We have tested this methodology in the nitrogen-fixing bacterium Azospirillum by amplyfing the total DNA extracted from several Azospirillum strains. We have used amplification bands with different specificity as molecular probes in hybridization experiments performed on amplified DNA. Results obtained have demonstrated the usefulness of this methodology for Azospirillum. Its use in microbial ecology studies as a general strategy to generate specific DNA probes is also discussed.

RAPD fingerprinting is useful for identification of Azospirillum strains.

In vitro amplification of genomic DNA fragments with single primers of arbitrary sequence was used as a rapid and sensitive method to obtain fingerprints of ten strains belonging to three of the Azospirillum species: brasilense, lipoferum and amazonense. Each strain showed a distinctive pattern of bands that permitted its unequivocal identification. Closely related strains produced almost identical fingerprints. Pairwise comparison and cluster analysis of the amplification patterns allowed grouping of the strains. The resulting dendrograms are similar to previous dendrograms based on the restriction endonuclease analysis (REA) of total DNA and on the restriction fragment length polymorphism (RFLP). Our results indicate that the random amplified polymorphic DNA (RAPD) technique is a simple, fast and useful tool for the determination of genetic relationships among Azospirillum isolates and to evaluate the genomic stability of the Azospirillum strains released in the environment.