Pandemic Swine flu

When a pandemic infection happens, the World Health Organization defines the extent of the virus’s spread; coordinates the international public-health response; selects the pandemic vaccine strains and recommends when large-scale vaccine production should start; and coordinates the gathering of scientific data on the outbreak.
WHO uses a six-points scale to define an infection, and only when significant outbreaks are achieved in one or two countries of WHO region and then virus spreads in other countries, is defined pandemic. Is swine flu a pandemic infection? Yes, in May 21th Margaret Chan described swine flu as level six: pandemic. She reiterated also the definition of a pandemic: “A defining characteristic of a pandemic is the almost-universal vulnerability of the world’s population to infection. Not all people become infected, but nearly all people are at risk.”
Not only geographical localization, but also severity is now considered in the definition of pandemic. Nevertheless, it is quite difficult to quantify worldwide one disease’s severity, because of different conditions in rich and poor countries, in terms of hospitals, number of medical doctors, drugs availability. Anyway, it’s right to call pandemic what is pandemic in order to move politicians and government to choose the better way to fight the infection.

Malaria vaccine soon on the market

In 2009 malaria vaccine enters in clinical phase III after a long term clinical study performed in Tanzania. Thousands of children die everyday in Sub-Saharan Africa because of malaria and men and women spend a lot of time and money to care this disease. The vaccine should be a great promise and hope for these populations.
The clinical trial of malaria vaccine involves 16000 children aged from two years to eleven in seven different African countries. The vaccine is a fusion of a protein from malaria parasite and surface antigen form hepatitis B virus. The main goal is block symptoms of malaria (fever, weakness, death) and maintain protection for several years. If everything goes well, vaccine will be submitted for regulatory approval by 2011 and it will be on the market by 2012. The Global Fund to Fight AIDS, Tuberculosis and Malaria and the Global Alliance for Vaccine and Immunizations ensure vaccine to reach the market.
This clinical trial open new opportunities to care malaria. This terrible disease kills a lot of children in poor countries every year also because anti-malaria drugs are very expensive and government cannot pay them. We have to keep our finger crossed for this clinical trial!

New approaches for animal model generation

Since 1880, when Louis Pasteur started his experiments demonstrating that sheep inoculated with anthrax bacteria survived if previously treated with his vaccine, experimentation with animals has been performed obtaining important results for scientific advances. Other examples can be done to describe the use of animals in scientific research: in 1920 dogs with diabetes were injected with extract of pancreatic cells and for the first time the role of insulin to care this disease was demonstrated. Two years ago, Mario Capecchi, Martin Evans and Oliver Smithies won the Nobel prize for the development of gene targeting techniques, important to generate transgenic animals.

Gene targeting is generally used to destroy a gene, producing knock out phenotype, to insert marker genes in order to understand gene regulation in particular genome region, or to alter cis-regulatory elements of the gene of interest. This technique was successfully applied in cultured mouse embryonic stem cells, and mice became the most common transgenic animals used in biomedical research. Nevertheless, mice are not the best model to understand all human diseases and in some cases transgenic mice failed to replicate human phenotype due to species-specific differences in physiology, cell biology or biochemistry. For this reason, great interest is now in the generation of non-rodent models, for instance pigs, monkeys, ferrets and also zebrafish. At the moment, the principal limitation is the availability of embryonic stem cells from these animals that haven’t been isolated yet.

Thus, alternative methods for gene targeting have recently been proposed. The first one uses recombinant adeno-associated virus (rAAV) as a vector to efficiently insert site-direct mutations in pig and ferret. AAV is a helper-dependent parvovirus, small and single DNA- stranded. Its genome presents inverted terminal repeats with an ordered secondary structure. DNA repair enzymes recognize these regions and allow recombination events. By inserting homologous sequences at both sites of the construct of interest, it’s possible to mutagenize specific genes.
Fibroblast are usually infected by rAAV, then their nucleus is used in somatic cell nuclear transfer (SCNT). The second approach introduced site-direct mutations in zebrafish genome. In this case, mutagenesis occurs through a Zinc-finger nuclease (ZFN), a chimeric protein consisting of Fok1 non specific endonuclease and 3/4 Zinc finger that bind specific DNA triplets. To work, Fok1 dimerizes and the presence of appropriate zinc finger domains guarantees high specificity. mRNA encoding ZFN is directly injected into single cell embryo.
Both these techniques improve genetic manipulation of the genomes of several species, such as pig, ferret, zebrafish and obviously mouse, allowing to produce new models for human diseases. Choosing the correct model is fundamental to obtain consistent results. In 1956 thalidomide experimentation was performed in wrong animal model, the drug wasn’t tested during pregnancy and embryotoxicity wasn’t observed. Everyone knows the terrible consequences of this unforgivable mistake.

Standardization in animals experiments

Today we would like to discuss about an important deal of experimental research: standardization procedure in animal experiments. Animal models are crucial to study human diseases but ethical problems concerning their use in lab are usually encountered. The US animal care and use regulations require scientists not to duplicate experiments previously performed in other labs, assuming that standardized procedures described in textbooks guarantee reproducibility between laboratories and make useless repetition.

These standardization procedures want to define environmental conditions in order to render animals homogeneous within an experiment and to improve comparability between labs. Is it possible to overcome all differences between labs? Can we obtain the same environmental conditions in winter or summer, for instance? Moreover, animals are sensible to noise, to room architecture and specially to the scientist that manipulate them. For this reason scientists often state in their publications that results only hold for the conditions under which the experiment has been carried out. In this case, generalization is not recommended and sometimes comparison with other works doesn’t make sense.
Other approaches to standardize experiments have to be proposed. We invite you to read an interesting paper in Nature Methods where authors proposed environmental heterogenization , a systematic way to control variation in experimental conditions. Good reading!

Signalling pathways as network of functional modules

Signalling pathways are usually described as a chain of consequential events, where proteins interact and transfer signal from external environment to nucleus. If some mutation occurs, all pathway is deregulated and it’s important to know where the mutation is in order to choose the correct approach to normalize it.

Is it possible to understand where is the mutation from genomic signature? It is! In Nature Reviews Genetics of June 2009 a new vision of signalling pathways is presented. Instead of a chain of reactions, functional modules are considered on basis of gene expression signature. From NCI-60 database authors identified genes related to the core protein of the pathway that showed the same variation of expression as this core gene.

For each pathway/ core protein several signatures have been identified and by relating signatures to mutants that selectively activate a specific downstream effector, it has been possible to assign each functional module (core protein and downstream effector) to genomic signature.

Which are the therapeutic implications of this new approach? For instance, it’s possible to define where is the mutation directly from genomic analysis, in this way a correct therapeutic approach can be chosen. Now, we have to increase our knowledge about the relationship between genomic signature and signalling pathway.

The Activity based protein profiling

About 30% of human proteins have not been characterized yet in terms of activity and substrates, but their incorrect functionality is often one molecular basis for human diseases.
A research group of The Scripps Research Institute tried to overcome this problem and developed a system, usable in high throughput screening, to analyse not well characterized proteins.

Their ABPP (Activity based protein profiling) approach uses a chemical probe enable to fit in catalytic site of a broader range of proteins, belonging to the same family. In this way it is negligible to know all biochemical properties of the specific protein or the ideal substrate for each target. The probe is conjugated to a polarized fluorophore that could monitor the change in the catalytic site conformation in presence of the inhibitor and make possible to use this technique in high throughput screening.
One limitation of this method is the need, at least in high throughput, for purified proteins, otherwise with such a generic substrate it could be difficult to relate results of a screening to a specific protein. In this case it’s crucial to retest positive hits in gel based assays in order to obtain straightforward data. However, ABPP represents also a good starting point for biochemical characterization of unknown proteins.

New method to quantify rare single-nucleotide polymorphism

Recent studies correlate rare allelic variants to many complex traits and combined effects of this deleterious mutations could explain susceptibility to many common diseases. To identify rare variants it’s necessary to genotype high number of individuals, by sequencing, or a pooled sample to minimize costs.

Druley and co-workers proposed a combination of molecular biology and computational analysis to achieve targeted resequencing and rare-variant detection. Procedure required PCR-amplification and sequencing with Illumina. They found that the first 12 bases of each Illumina read contained significantly fewer errors than later and, so, they used only this portion for their analysis.
They developed a new algorithm based on large deviation theory, named SNPSeeker. This program uses a second-order dependency error model for single-nucleotide polymorphism identification and considers the position of sequencing read (PCR cycle number) and the identity of two upstream bases. Thus, they improved the specificity of SNP calling and obtained results comparable to Sanger sequencing data, consistently reducing costs.
An important application of this method, is the combination of pooled-sample sequencing with genomic selection strategy to perform a more systematic survey of protein-coding DNA. This knowledge would be an important achievement for disease screening and tailoring risk-appropriate therapy.

Small molecule inhibitors and resistance

A great challenge in academic labs and pharmaceutical industries is the identification of small molecule inhibitors that could specifically target proteins involved in human diseases.
To do this, the most important step is the knowledge of biochemical features of the target protein. Human diseases associated to the target are usually well understood and the target has been previously validated: this means that its inhibition determines a recovery of normal cellular functionality.

X-ray crystallographic analysis and SAR (structure activity relationship) studies are crucial to identify and improve small molecules; otherwise, high throughput screening are performed, this route is usually followed by pharmaceutical or biotech companies that dispose of instrumentation to run the screening and manage data. Several small molecules identified as potential drug in one disease are tested in clinical studies in order to bypass resistance problems that could arise during long term treatment. Indeed, during this therapies a natural selection of mutations that provide the restoring of protein target activity, happens and makes useless the treatment.
Nevertheless, it’s necessary to continue research of small molecule inhibitor, especially in view of good results obtained, for instance, in tumoral diseases.

Regenerative medicine

In latest years, worldwide research is focussed on stem cells and their potentiality to regenerate tissues upon specific stimuli. Researchers from the Stanford University implemented a great system to culture adult stem cells.

They used microcirculatory beds as a life support. These beds presented one afferent artery, one efferent vein and capillary bed, surrounding parenchymal tissue, in order to mimic vassal circulation. A special bioreactor that provided oxygen and nutrients was used to maintain alive tissue.
Then, they seeded different kind of stem cells and in all cases they observed a migration of cells from vessels to parenchymal tissue and cluster formation. This means that with this support adult stem cells can survive, replicate and in the next future produce replacement organs.
Using autologous microcirculatory beds explanted from host vessels, researchers overcame all problems related to reimplatation into host circulation. By transfecting luciferase gene into seeded stem cells, they followed viability of stem cells also in vivo.
This work seems really promising: for example to restore organ functionality it is enough to seed beds with stem cells producing insulin, clotting factors or other signalling proteins to make possible a complete recovery of physiological status. Anyway, this is only a starting point!

SIOPEN-R-NET and the role of Internet in clinical trials

SIOPEN is the European Neuroblastoma Group of the International Society for Paediatric Oncology dedicated to study and care neuroblastoma. They organize a web space in which exchange acquired experiences: for instance, a tumour bank is present in order to determine prognostic parameters by biological studies conducted in several labs or hospitals as well as central serum banking allows pharmacokinetic studies to correlate drug levels with efficacy or toxicity in order to determine the optimum doses and improve medical procedures.

Moreover, SIOPEN-R-NET disposes on central database in which electronic case report forms (eCRFs), electronic data capture, remote randomisation and the distribution of information on trial progress are available. This network is currently used by 345 active users from 250 institutions in 18 European countries and gives also an important support to associations of families with children suffering from this disease. SIOPEN-R-NET is one example of how IT could help science to advance and medical programs to improve.

Thus, Internet is also an useful tool to make easily accessible, secure, safe and rapid multi-institutional clinical trials: geographically distant centers can enrol patients, promptly capture and manage data and communicate their results in real time, reducing paper chart documentation.