In this research summary of two new studies from Blizard Institute researchers, we hear about new ways to boost the innate immune function of Hepatitis B patients that could shorten treatments, and serious questions are raised on current treatment decisions for young patients.

1) Interferon alpha induces sustained changes in NK cell responsiveness to hepatitis B viral load suppression in vivo. PLoS Pathog. (2016)

In this work, supervised by Dr Patrick Kennedy (Reader in Hepatology & Honorary Consultant, QMUL) and Professor Mala Maini (Wellcome Trust Senior Investigator & Professor of Viral Immunology at UCL), the research team observed novel responses to hepatitis B treatment.

As first author, Dr Upkar Gill, funded by a Wellcome Trust Clinical Research Training Fellowship, performed the experimental work using carefully selected samples from patients treated at The Royal London Hospital.

What is new about the study?

  • The current treatments for hepatitis B, interferon and oral antivirals, act differentially on the immune response.
  • The team shows that oral antivirals given in sequence, after interferon (sequential therapy) maintains boosting of the innate immune response (NK cell function).
  • Loss of HBsAg (the surface antigen of the hepatitis B virus) is the ideal curative outcome. HBsAg level reduction is greater with sequential therapy than other therapies alone, highlighting the potential benefit of interferon priming.
  • This sustained boosting of the innate immune response following interferon priming has not previously been described.

How was the study carried out?

  • The study was carried out using peripheral blood samples from patients undergoing treatment at The Royal London Hospital.
  • Immune cells from the blood were analysed to determine their composition and function and these were correlated with the clinical outcomes for the treated patients.

Is there anything surprising about the results?

  • There is limited data on immune cell function in patients treated for hepatitis B.
  • The continued boosting of NK cells is surprising as they are thought to be ‘short lived’ cells, but the data show they may have the potential of maintaining a ‘long lived’ population with antiviral function, important for hepatitis B clearance.

Why is the study important?

  • The findings in this study provide a mechanistic rationale for using interferon/oral antivirals in sequence or in combination to improve treatment outcomes in hepatitis B.
  • The improved reduction of HBsAg with this treatment strategy could reduce the duration of treatments, which is an important endpoint in hepatitis B therapy.

 

What are the wider implications?

  • The results from this study provide a springboard for larger clinical trials to explore the value of shorter courses of interferon, to prime the immune response.
  • Definitive treatment endpoints and reductions in HBsAg, ultimately leading to a cure have wide implications to reduce complications of chronic liver disease and liver cancer on a global scale.
  • The potential of shorter treatment durations would mean that younger patients could be treated, without the burden of life long therapy thus increasing the pool of patients for treatment candidacy.

 

 

2) HBV DNA Integration and Clonal Hepatocyte Expansion in Chronic Hepatitis B Patients Considered Immune Tolerant, Gastroenterology (2016)

In this work, conceived by Dr Patrick Kennedy and international collaborators Professor Mason (Philadelphia, USA) and Professor Bertoletti (Singapore) novel findings challenging the concept of ‘immune tolerant’ chronic hepatitis B (CHB) are presented, which challenge the premise on which current treatment decisions are made. Drs Upkar Gill and Patrick Kennedy, have been studying the evolution of CHB in children and young adults from the dedicated clinics at The Royal London Hospital. Using valuable blood and liver tissue samples from these patients, collaborating with other key leaders in the field, this work has led to the generation of novel findings in the management of the disease in young people. Current national and international treatment guidelines exclude young patients (believed to be in this immune tolerant phase) from treatment, but the new results raise serious questions about this approach.

In line with previous data, (http://www.gastrojournal.org/article/S0016-5085(12)00840-2/fulltext) the team observed that young patients considered to have “immune tolerant” CHB, in fact have evidence of immunopathology and the potential for progressive disease, no different to their peers with “active” disease.

What is new about the study?

  • Chronic Hepatitis B (CHB), progresses through disease phases; the first of which, the ‘immune tolerant’ phase is thought to be a disease-free state and thus patients are not considered for therapy.
  • In keeping with previous work, (http://www.gastrojournal.org/article/S0016-5085(12)00840-2/fulltext) the team shows that there is evidence of immunopathology in young patients considered immune tolerant.
  • Importantly, they also demonstrate these young patients have evidence of HBV DNA integration and clonal expansion of hepatocytes; events which could lead to the development of liver cancer over time.

How did you carry out the study?

  • The study was carried out using peripheral blood samples and liver biopsy tissue specimens from children and adults managed in The Royal London Hospital Viral Hepatitis Service.
  • Immune cells from the blood were analysed to determine T cell function and liver tissue was analysed for DNA integration, clonal expansion of liver cells and expression of viral proteins in liver tissue.

Is there anything surprising about the results?

  • Data on immune function in CHB patients considered immune tolerant is limited.
  • Historically, these young patients were considered to have mild disease, devoid of disease progression. Conversely, our data is at odds with this perception and in fact demonstrates that these patients already have progressive disease and therefore are already at risk for the development of advanced liver disease and liver cancer.

Why is the study important?

  • CHB is the leading cause of primary liver cancer worldwide
  • This study makes a compelling case that historical terms, such as “immune tolerance”, are misleading and challenges the clinical categorisations on which treatment decisions are made.
  • Events leading to the development of liver cancer may already be present in young CHB patients.
  • Young patients, previously excluded from treatment, may benefit from early antiviral therapy, which potentially could prevent disease progression and the development of liver cancer.

What are the wider implications?

  • The term immune tolerant CHB should not be used to define the early phase of CHB infection as it is both inaccurate and out-dated. Treating physicians should now describe this phenotype as the “high replication, low inflammation stage” and importantly, should be more circumspect about treatment and management of these patients.
  • These results should provide a springboard for larger studies to evaluate the potential therapeutic benefits of early treatment in CHB at a time of major change in the HBV treatment landscape.
  • Adjusting the bar for treatment candidacy may mean that these patients would be treated earlier to prevent advanced liver disease and liver cancer complicating CHB infection.

Missing link in genetics could explain conundrum of disease inheritance - Research by Prof Vardhman Rakyan and his group published in Science magazine

The process by which a mother’s diet during pregnancy can permanently affect her offspring’s attributes, such as weight, could be strongly influenced by genetic variation in an unexpected part of the genome, according to research led by Prof Vardhman Rakyan from the Centre for Genomics and Child Health.

                   Epigenetics: image credit Christoph Bock

The discovery could shed light on why many human genetic studies have previously not been able to fully explain how certain diseases, such as type 2 diabetes and obesity, are inherited.

The study, published in Science and co-authored by University of Cambridge and King’s College London, shows that the genetic variation of ribosomal DNA (rDNA) could be driving how the environment within the womb determines an offspring’s attributes. rDNA is the genetic material that forms ribosomes - the protein building machines within the cell.

Lead researcher Professor Vardhman Rakyan: “The fact that genetic variation of ribosomal DNA seems to play such a major role suggests that many human genetics studies could be missing a key part of the puzzle. These studies only looked at a single copy part of individuals’ genomes and never at ribosomal DNA.

“This could be the reason why we’ve only so far been able to explain a small fraction of the heritability of many health conditions, which makes a lot of sense in the context of metabolic diseases, such as type 2 diabetes.”

The environmental factors that play a role alongside genetic factors in determining a person’s attributes are also present in the in-utero environment. When offspring are in the womb, what their mothers experience environmentally (for example, diet, stress, smoking), influences the attributes of offspring when they are adults. This ‘developmental programming’ is understood to be a large contributor to the obesity epidemic seen today.

A major contributor to this process is ‘epigenetics’. This describes naturally-occurring modifications to genes that control how they are expressed. One such modification involves tagging DNA with chemical compounds called methyl groups. These epigenetic markers determine which genes are expressed or not expressed. Liver cells and kidney cells are genetically identical apart from their epigenetic marks. It has been proposed that in response to a poor in-utero environment, an offspring’s epigenetic profile will change.

The team compared the offspring of pregnant mice when given a low protein diet (8 per cent protein) and a normal diet (20 per cent protein). After they were weaned, all offspring were given a normal diet, and the team then looked at the difference in the offspring’s DNA methylation, from mothers exposed to low protein and those that were not.

Professor Rakyan said: “Initially, we found nothing, so that was a big surprise, but then we looked at the data in a different way. We looked at the ribosomal DNA data and found huge epigenetic differences.

“When cells are stressed, for example when nutrient levels are low, they alter protein production as a survival strategy. In our low protein mice mothers, we saw that their offspring had methylated rDNA. This slowed the expression of their rDNA, which could be influencing the function of ribosomes, and resulted in smaller offspring – as much as 25 per cent lighter.”

These epigenetic effects occur in a critical developmental window while the offspring is in-utero but is a permanent effect that remains into adulthood. A mother’s low protein diet while pregnant is therefore likely to have more severe consequence on the offspring’s epigenetic state and weight than an offspring’s own diet after it has been weaned.

Professor Rakyan added: “Looking beyond the epigenetic markers, when we looked at the basic genetic sequence of the rDNA, we found an even bigger surprise. Even though all the mice in the study were bred to be genetically identical, we found that the rDNA between the individual mice was not genetically identical, and that even within an individual mouse, different copies of rDNA were genetically distinct. So there is huge variation in rDNA which is also playing a big role in determining the attributes of offspring.”

In any given genome, there are many copies of rDNA, and Professor Rakyan and colleagues found that not all copies of the rDNA were responding epigenetically. In offspring from mothers who were fed on low protein diets, it was only one form of rDNA – the ‘A-variant’ - that appeared to undergo methylation and affect weight.

This means that the epigenetic response of a given mouse is determined by the genetic variation of the rDNA - those who have more A-variant rDNA end up being smaller.

Heritability (how much the risk of a disease is explained by genetic factors) of type 2 diabetes has been estimated to be between 25 and 80 per cent in different studies. However, only about 20 per cent of the heritability of type 2 diabetes has been explained by genome studies of people with the disease. The major role that genetic variation of rDNA appears to have and the fact that rDNA analysis would not have been included in these studies could explain some of this missing heritability.

The findings also complement other studies that have found that mice that are put on high fat diets have offspring who show increased rDNA methylation. This suggests that methylation is a general stress response and may also explain the rise in obesity that is happening across the world.

The study was funded by the Biotechnology and Biological Sciences Research Council (BBSRC), Research Councils UK, EU-FP7, British Heart Foundation and Medical Research Council (MRC).

More information

CANBUILD - Deconstructing Cancer is a £2 million project funded by the European Research Council and lead by Prof Francis Balkwill, which aims to revolutionise the field of cancer cell research using bioengineering techniques to grow the first complex 3D human tumour in the laboratory.

The CANBUILD team will exhibit at the Royal Society's Summer Science Fair from Monday 4th July to Sunday 10th July in London. For more information on the fair CLICK HERE.

The project is a collaboration of scientists from across Queen Mary University of London, from the Blizard Institute, Barts Cancer Institute and the School of Engineering and Materials Science.

Human cancers are more than just malignant cells, even at their very beginning. Other cells and chemicals that normally support and protect the body are hijacked by the cancer cells, instead helping the cancer to grow and spread.

Recent research has shown us the importance of targeting not only the malignant cancer cells but also this wider tumour microenvironment for long-term cancer treatment.

In the CANBUILD project, the multi-disciplinary team of scientists will be using the latest advances in tissue engineering, biomechanics, imaging and stem cell biology. They believe this combination will make it possible to engineer, for the first time, a complex 3D human tumour in which the different cell types making up the tumour microenvironment could communicate, evolve and grow in vitro (outside the body, in the laboratory).

The CANBUILD goal is to recreate the tumour microenvironment found in high-grade human ovarian cancer; the subtype that leads to 70 per cent of all ovarian cancer deaths. However, the research may have implications for several other cancer types as well.

The vision is that this project will replace traditional techniques in which human cancer cells are grown in isolation on plastic surfaces. Success in the CANBUILD project may also provide better ways of testing new drugs that target the human tumour microenvironment.