Researchers show existence of eye microbiome

Bugs in your eyes may be a good thing. Resident microbes living on the eye are essential for immune responses that protect the eye from infection, new research shows. The study, which appears in the journal Immunity on 27 June 2017, demonstrates the existence of a resident ocular microbiome that trains the developing immune system to fend off pathogens. The research was conducted at the US National Eye Institute (NEI), part of the National Institutes of Health.

“This is the first evidence that a bacterium lives on the ocular surface long-term,” explained Rachel Caspi, Ph.D., senior investigator in NEI’s Laboratory of Immunology.

“This work addresses a longstanding question about whether there is a resident ocular microbiome.”

For years, the ocular surface was thought to be sterile because of the presence of an enzyme called lysozyme that destroys bacteria, antimicrobial peptides, and other factors that rid the eye of microbes that may land from the air (or from our fingers) onto the surface of the eye.

Anthony St. Leger, Ph.D., research fellow in Caspi’s laboratory, was able to culture bacteria from the mouse conjunctiva, the membrane that lines the eyelids. He found several species of Staphylococci, which are commonly found on the skin, and Corynebacterium mastitidis (C. mast). But it wasn’t clear whether those microbes had just arrived on the eye and were en route to being destroyed, or whether they lived on the eye for extended periods of time.

The researchers found that C. mast, when cultured with immune cells from the conjunctiva, induced the production of interleukin (IL)-17, a signalling protein critical for host defence. Upon further investigation, they found that IL-17 was produced by gamma delta T cells, a type of immune cell found in mucosal tissues. IL-17 attracted other immune cells called neutrophils -- the most abundant type of white blood cell -- to the conjunctiva and induced the release of anti-microbial proteins into the tears. The researchers are currently investigating the unique features that can make C. mast resistant to the immune response that it itself provokes and allow it to persist in the eye.

To determine whether the microbe was contributing to the immune response in mice, St. Leger formed two groups, one control (with C. mast) and one treated with an antibiotic to kill C. mast and other ocular bacteria, and then challenged them with the fungus, Candida albicans. The mice receiving antibiotics had a reduced immune response in their conjunctiva and were not able to eliminate C. albicans, leading to fullblown ocular infection. The control mice with normal C. mast on the other hand were able to fend off the fungus.

St. Leger noticed that mice from the NIH animal facility had C. mast on their eyes, but mice from the Jackson Laboratory (JAX) in Maine and other commercial vendors did not. This fortuitous observation allowed the researchers to determine if C. mast was truly a resident microbe, as opposed to a transient microbe that lands on the eye from the environment. They did this by inoculating C. mast-free mice with the microbe and determining if the microbe could be cultured from those animals’ eyes many weeks later. They also determined whether the microbe could easily be transmitted among cagemates.

When inoculated with C. mast, JAX mice produced conjunctival gamma delta T cells that released IL-17. Bacteria could still be cultured from their eyes after many weeks. By contrast, several other strains of bacteria inoculated onto the eyes of JAX mice disappeared without inducing local immunity. “We still don’t know what enables C. mast to successfully establish itself in the eye, whereas other similar bacteria fail to colonize,” Caspi said.

Interestingly, C. mast was not spread to cage-mates even after eight weeks of cohousing; however, C. mast can be passed from mother to pup. Both of these observations support the notion that C. mast is a resident commensal, not a bacterium that is continually re-introduced to the eye from the skin or the environment, Caspi explained.

Although C. mast appears to stimulate a beneficial immune response, there may be situations in which it could cause disease, St. Leger noted. For instance, the elderly tend to have suppressed immune systems, which might allow C. mast to grow out of control and cause disease.

The researchers are currently investigating whether other bacteria play a role in regulating eye immunity.

“We’ve established the proof of concept of a central ocular microbiome,” St. Leger said. “It’s well known that there are good bacteria in the gut that modulate the immune response. Now we show that this relationship exists in the eye. That’s important for how we think about treating ocular disease.”

  • doi: 10.1016/j.immuni.2017.06.014

Biological bypass shows promise in coronary artery disease

A new gene therapy that targets the heart and requires only one treatment session has been found safe for patients with coronary artery disease, according to a successful trial carried out in Finland. Enhancing circulation in the oxygen-deficient heart muscle, the effects were visible even one year after the treatment.

The randomised, blinded, placebo-controlled phase 1/2a trial was carried out in collaboration between the University of Eastern Finland, Kuopio University Hospital andTurku PET Centre as part of the Centre of Excellence in Cardiovascular and Metabolic Diseases of the Academy of Finland.

The biological bypass is based on gene transfer in which a natural human growth factor is injected into the heart muscle to enhance vascular growth. The trial was the first in the world to use a novel vascular growth factor that has several beneficial effects on circulation in the heart muscle. The trial also developed a novel and precise method for injecting the gene into the oxygen-deficient heart muscle area. A customised catheter is inserted via the patient’s groin vessels to the left ventricle, after which the gene solution can be injected directly into the heart muscle. The method is as easy to perform as coronary balloon angioplasty, which means that it is also suitable for older patients and patients who are beyond a bypass surgery or other demanding surgical or arterial operations.

The biological bypass constitutes a significant step forward in the development of novel biological treatments for patients with severe coronary artery disease. A new blood test biomarker was also discovered, making it possible to identify patients who are most likely to benefit from the new treatment.

The biological bypass was developed by Academy Professor Seppo Ylä-Herttuala’s research group at the A.I. Virtanen Institute for Molecular Sciences of the University of Eastern Finland. At the Kuopio University Hospital Heart Centre, Professor Juha Hartikainen was responsible for the trial.

  • doi: 10.1093/eurheartj/ehx352

Researchers identify the component that allows a lethal type of bacteria to spread resistance to antibiotics

Antibiotic resistance of the bacterium Staphylococcus aureus is responsible for 11,300 deaths a year in the United States alone – a figure that corresponds to half of all deaths caused by gram-positive resistant bacteria in that country. Such high mortality is related to the speed at which the bacterium acquires resistance to antibiotics. A study performed at the Institute for Research in Biomedicine (IRB Barcelona) and involving the collaboration of the Centro de Investigaciones Biológicas (CIB-CSIC) in Madrid has identified the key component of the machinery that S. aureus uses to acquire and transfer genes that confer resistance to antibiotics. The work has been published this week in the Proceedings of the National Academic of Sciences (PNAS).

“The battle against bacteria – particularly in the hospital setting where they are a major threat – implies understanding how genes are transferred to adapt to a changing environment. For example, when they are treated with new antibiotics,” explains the head of the study and IRB Barcelona group leader Miquel Coll, also a CSIC researcher, who studies horizontal gene transfer from a structural biology perspective.

“Horizontal gene transfer confers bacteria with an extraordinary capacity to evolve and adapt rapidly – a capacity that humans do not have for example,” says Coll. One of these pathways is called conjugation, a process by which two bacteria join and one of them transfers a piece of DNA called plasmid to the other. “A plasmid is a small piece of circular DNA that holds very few genes, often including those for antibiotic resistance and it takes only a few minutes to be passed between bacteria,” he explains.

Horizontal gene transfer involves machinery in which the relaxase, an enzymatic protein, is a key component. Thanks to the 3D resolution of the structure of the complex formed by the relaxase with a fragment of the plasmid DNA, the researchers have identified that an amino acid histidine is a pivotal element in the DNA processing and thus in the transfer and the spread of resistance. “What we have discovered is that the relaxase of diverse strains of S. aureus differs because it uses an amino acid that is not used by any other relaxase that we know of,” explains the first author of the study, Radoslaw Pluta, former “la Caixa” PhD student at IRB Barcelona, and currently a postdoctoral researcher at the International Institute of Molecular and Cell Biology in Warsaw, Poland.

Histidine is the catalytic residue that allows the relaxase to cut DNA, bind to it, and stretch one of the two strands and take it into the receptor bacterium, where the strand replicates to form a double strand of the plasmid again. This new plasmid now holds the resistance genes and the machinery to transfer them to another bacterium. The scientists indicate that this catalytic histidine is present in the relaxases of 85% of the strains of Staphylococcus aureus.

To test whether histidine is decisive in horizontal gene transfer, researchers in Manuel Espinosa’s group at the CIB-CSIC, who participated in the study, replaced it bya different amino acid and confirmed that it prevents transfer in culture dishes.

The mutation of histidine does not kill that bacterium but rather prevents gene transfer. How could this mechanism be exploited to fight infections? “I don’t know,” says Coll, “but we now know more details about a lethal bacterium and this may pave the way to the development of molecules to prevent the spread of resistant strains.”

Coll explains that hospital infections are the most difficult types to tackle. “We are in a race that we always lose because when a new antibiotic is brought out, resistance quickly emerges and spreads,” he describes. The scientist adds that the list of antibiotics for hospital use is “too” short. Apart from the difficulty involved in developing new antibiotics, Coll also comments on another obstacle impeding advancement. “There is little investment because the pharmaceutical industry has other priorities. While this is perfectly valid, resources from the public and private sectors should be pooled.”

This work has involved the collaboration of Modesto Orozco’s group, also at IRB Barcelona, which has performed the theoretical studies to validate the chemical reaction between the plasmid DNA and the protein via histidine. The structural resolution of the complex formed by the protein and the DNA has been achieved using data obtained by X-ray diffraction at the European synchrotron in Grenoble.

  • doi: 10.1073/pnas.1702971114

Study identifies essential genes for cancer immunotherapy

A new study identifies genes that are necessary in cancer cells for immunotherapy to work, addressing the problem of why some tumours don’t respond to immunotherapy or respond initially but then stop as tumour cells develop resistance to immunotherapy.

The study, from the US National Cancer Institute (NCI), was led by Nicholas Restifo, M.D., a senior investigator with NCI’s Center for Cancer Research, with coauthors from NCI; Georgetown University, Washington D.C.; the Broad Institute of MIT and Harvard University, Cambridge, Massachusetts; New York University, New York City; and the University of Pennsylvania, Philadelphia. It was published online in Nature on 7 August 2017.

“There is a great deal of interest in cancer immunotherapy, especially for patients who have metastatic cancer,” said Dr Restifo. “The response to immunotherapy can be fantastic, but understanding why some patients don’t respond will help us improve treatments for more patients.”

Cancer immunotherapy relies on T cells, a type of cell in the immune system, to destroy tumours. Dr Restifo and his colleagues have previously shown that the infusion of large numbers of T cells can trigger complete regression of cancer in patients. They and others have also shown that T cells can directly recognize and kill tumour cells.

However, some tumour cells are resistant to the destruction unleashed by T cells. To investigate the basis for this resistance, the researchers sought to identify the genes in cancer cells that are necessary for them to be killed by T cells.

Working with a melanoma tumour cell line, the researchers used a gene editing technology called CRISPR that “knocks out,” or stops the expression, of individual genes in cancer cells. By knocking out every known protein-encoding gene in the human genome and then testing the ability of the gene-modified melanoma cells to respond to T cells, they found more than 100 genes that may play a role in facilitating tumour destruction by T cells.

Once the team identified these “candidate” genes, they sought additional evidence that these genes play a role in susceptibility to T cell-mediated killing. To this end, they examined data on “cytolytic activity,” or a genetic profile that shows cancer cells are responding to T cells, in more than 11,000 patient tumours from The Cancer Genome Atlas, a collaboration between NCI and the National Human Genome Research Institute. They found that a number of the genes identified in the CRISPR screen as being necessary for tumour cells to respond to T cells were indeed associated with tumour cytolytic activity in patient samples.

One such gene is called APLNR. The product of this gene is a protein called the apelin receptor. Although it had been suspected to contribute to the development of some cancers, this was the first indication of a role in the response to T cells. Further investigation of tumours from patients resistant to immunotherapies showed that the apelin receptor protein was nonfunctional in some of them, indicating that the loss of this protein may limit the response to immunotherapy treatment.

Shashank Patel, Ph.D., the first author of the study, said the results show that “many more genes than we originally expected play a vital role in dictating the success of cancer immunotherapies.”

The researchers wrote that this gene list could serve as a blueprint to study the emergence of tumour resistance to T cell-based cancer therapies. Dr Restifo noted that if this set of genes is validated in clinical trials, then this data could eventually lead to more effective treatments for patients.


Vaccine shows protection against gonorrhoea

Exposure to the meningococcal group B vaccine during a New Zealand mass vaccination campaign was associated with a reduced likelihood of contracting gonorrhoea, compared with unvaccinated people, according to a new study of more than 14,000 people published in The Lancet. This is the first time that a vaccine has shown any protection against gonorrhoea, and may provide a new avenue for vaccine development.

If the effect is confirmed in other currently available and similar meningococcal group B vaccines, administering the vaccine in adolescence could result in significant declines in gonorrhoea, which has increasingly become drug resistant.

The importance of a vaccine candidate that may have even a moderate effect on reducing rates of infection is highlighted in a new report by The Lancet Infectious Diseases journal which urges global policy action to address sexually transmitted infections (STIs).

So far, efforts to develop a vaccine against gonorrhoea have been unsuccessful despite more than a century of research. Four vaccine candidates have reached clinical trial stage but none have been effective. However, population data suggests there is a decline in gonorrhoea immediately after the use ofthe outer membrane vesicle (OMV) meningococcal group B vaccine in Cuba, New Zealand, and Norway.

Despite the two diseases being very different in terms of symptoms and mode of transmission, there is an 80-90% genetic match between the Neisseria gonorrhoeae and Neisseria meningitidis bacteria, providing a biologically plausible mechanism for cross-protection.

The researchers note that because of the variability of different strains of N. gonorrhoeae and N. meningitidis bacteria, the effect of the vaccine might vary depending on the strain. And being co-infected with chlamydia slightly reduced the effectiveness of the vaccine.


Herpes virus study in mice leads to potential broad-spectrum antiviral

After herpesviruses infect a cell, their genomes are assembled into specialized protein structures called nucelosomes. Many cellular enzyme complexes can modulate these structures to either promote or inhibit the progression of infection. Scientists studying how one of these complexes (EZH2/1) regulated herpes simplex virus (HSV) infection unexpectedly found that inhibiting EZH2/1 suppressed viral infection. The research group, from the National Institute of Allergy and Infectious Diseases (NIAID) at the National Institutes of Health, then demonstrated that EZH2/1 inhibitors also enhanced the cellular antiviral response in cultured cells and in mice.

Once a person has been infected with a herpesvirus, the virus persists in a latent form, sometimes reactivating to cause recurrent disease. Two-thirds of the global population are infected with HSV-1, and at least 500 million are infected with HSV-2, according to the World Health Organization. These viruses cause a range of diseases and conditions from oral cold sores to genital lesions to serious eye infections that can lead to blindness. In infants who acquire the infection from their mothers, HSV can cause neurological and developmental problems. People infected with HSV also have an enhanced risk of acquiring or transmitting human immunodeficiency virus (HIV). Treatment usually involves antiviral drugs that interfere with viral replication, but new approaches to combat these infections are needed.

The NIAID group demonstrated that EZH2/1 inhibitors not only suppressed HSV infection, spread, and reactivation in mice, but also suppressed human cytomegalovirus, adenovirus, and Zika virus infections in cell culture using human primary fibroblast cell lines. These authors suggest that EZH2/1 inhibitors have considerable potential as broad-spectrum antivirals.

  • doi: 10.1128/mBio.01141-17 (2017)

New imaging technique overturns longstanding textbook model of DNA folding

How can six and half feet of DNA be folded into the tiny nucleus of a cell? Researchers have developed a new imaging method that visualizes a very different DNA structure, featuring small folds of DNA in close proximity. The study reveals that the DNAprotein structure, known as chromatin, is a much more diverse and flexible chain than previously thought. This provides exciting new insights into how chromatin directs a nimbler interaction between different genes to regulate gene expression, and provides a mechanism for chemical modifications of DNA to be maintained as cells divide.

For decades, experiments suggested a hierarchical folding model in which DNA segments spooled around 11 nanometer-sized protein particles, assembled into rigid fibres that folded into larger and larger loops to form chromosomes. However, that model was based on structures of chromatin in vitro after harsh chemical extraction of cellular components.

Now, researchers at the Salk Institute, La Jolla, California, funded by the NIH Common Fund, have developed an electron microscopy technique called ChromEMT that enables the 3D structure and packing of DNA to be visualized inside the cell nucleus of intact cells. Contrary to the longstanding text book models, DNA forms flexible chromatin chains that have fluctuating diameters between five and 24 nanometers that collapse and pack together in a wide range of configurations and concentrations.

The newly observed and diverse array of structures provides for a more flexible human genome that can bend at varying lengths and rapidly collapse into chromosomes at cell division. It explains how variations in DNA sequences and interactions could result in different structures that exquisitely fine tune the activity and expression of genes.

“This is groundbreaking work that will change the genetics and biochemistry textbooks,” remarks Roderic I. Pettigrew, Ph.D., M.D., director of the National Institute of Biomedical Imaging and Bioengineering (NIBIB), which administered the grant. “It’s an outstanding example of how constantly improving imaging techniques continue to show the true structure of everything from neuronal connections in the brain to the correct visualization of gene expression in the cell. It reveals how these complex biological structures are able to perform the myriad intricate and elaborate functions of the human body.”

“We identified a fluorescent small molecule that binds specifically to DNA and can be visualized using advanced new 3D imaging methods with the electron microscope,” explained Clodagh O’Shea, Ph.D., the leader of the Salk group, associate professor and Howard Hughes Medical Institute Faculty Scholar. “The system enables individual DNA particles, chains and chromosomes to be visualized in 3D in a live, single cell. Thus, we are able to see the fine structure and interactions of DNA and chromatin in the nucleus of intact cells.”

The researchers believe their discovery dovetails with their research on how tumour viruses and cancer mutations change a cell’s DNA structure and organization to cause uncontrolled cell growth. It could enable the design of new drugs that manipulate the structure and organization of DNA to make a tumour cell ‘remember’ how to be normal again or impart new functions that improve the human condition.

“To see the human genome in in all of its 3D glory is the dream of every biologist. Now, we are working to design probes that will allow us to also see the proteins that bind to the DNA to turn genes on and off. We will then be able to view an actual gene in action,” concluded O’Shea.

 

 

Date of upload: 22nd Sep 2017

                                  
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