Some patients at KwaZulu-Natal hospitals are donating lung tissue for use in TB research. Sue Segar tracks what happens to these tissue samples and explores why these samples are so important to unlocking the mysteries of what the TB bacterium does inside the human body.
It is Friday morning at the King Dinuzulu Hospital in Durban, KwaZulu-Natal. A tuberculosis (TB) patient with advanced lung disease has been booked for surgery to treat massive haemoptysis - the medical term for coughing up blood from the lungs.
Surgeons from the hospital's cardiothoracic team are preparing to operate. Ahead of the surgery, thanks to a unique, collaborative arrangement, they've alerted the clinical team at the Africa Health Research Institute (AHRI), also in Durban.
This is the cue for a team from AHRI to travel to the hospital to visit the patient ahead of the surgery, to discuss the research they are doing on TB, and to ask for permission to take a sample of the lung tissue once the surgery is completed. The team also asks for access to the patient's medical history in order to characterise the nature of the disease and its progression in this particular patient.
"The team that visits the TB patient is made up of nurses and other support staff specifically trained for this purpose," says Professor Threnesan Naidoo, research pathologist at AHRI. "There's absolutely no pressure for the patient to agree. But most do agree. You'd be surprised to know how much our public is supportive of TB research," says Naidoo, a recurrent TB survivor himself. "I believe this is simply because the people personally know the reality and devastation of TB. They want to get rid of it just as much as doctors and scientists do."
TB is a chronic disease that has been around for centuries. It is caused by a rod-shaped bacterium - mycobacterium tuberculosis - that mainly invades the lungs but can also spread to other parts of the body. The disease is typically contracted by breathing in droplets coughed up by people with TB. Once the organisms enter the lungs, they can cause disease immediately or can lie dormant in the tissues, sometimes for decades. How and why they persist in the human body is not fully understood.
Journey to the lab
"The surgery process follows the normal diagnostic workflow, and once it's done, the lung part that has been removed is put into formalin [a chemical that keeps the lung preserved for analysis] and it goes first to the state laboratory pathologist who looks at the specimen as they would any biopsy or surgical specimen. They make their diagnosis and give that to the clinician," explains Naidoo. "Once this has happened, our collaborators alert us - and from there, our processes are in place to retrieve what we call the reserve specimen - whatever's left over."
He adds: "We have dedicated space to store the tissue at AHRI, in a manner that is respectful to the tissue. We are constantly mindful that the tissue is from human beings." He says all the research they do goes through a stringent ethics review process done by the University of KwaZulu-Natal Biomedical Research Ethics Committee.
High tech research
The research process starts off with scientists looking at the samples with the naked eye. "There's so much you can tell about the disease just by looking at the tissue, and how it differs from that of a normal lung," says Professor Adrie Steyn, also from AHRI. "We also look at the tissue using a microscope, but that only gives a two-dimensional perspective. This is where our highly advanced technology comes in."
One of these advanced technologies is high-resolution micro-computed tomography - an imaging technique used to reconstruct detailed three-dimensional views of tissue. These detailed three-dimensional images of lung tissue are one of the things that enables the AHRI team to be at the cutting edge of this type of research. The highly specialised instrument they use for this costs around R15 million.
Cellular light shows
Another important research technique, called multiplex immunofluorescence, involves selectively letting certain cells light up and letting the resulting light show tell the story of what is happening between the cells.
To follow how this works, we first need to recap some basics. An antigen is an agent such as a bacterium or virus or other foreign molecule that triggers an immune response. One form of immune response is the production of antibodies. Antibodies are proteins made by the body specifically to 'attack' a particular antigen. They contain a code of amino acids - the building blocks of proteins - that compliment those of the antigen. Antibodies circulate in the bloodstream. When they come into contact with 'their' antigen, they form a complex by joining together at the complimentary sites in what is called a 'lock and key' fashion. From there, various processes take place to inactivate or kill the infectious agent.
Fortunately, antibodies can be made synthetically or in animals and then used as diagnostic or research tools in laboratories. For instance, with multiplex immunofluorescence, antibodies are modified by labelling them with fluorescent molecules. In the case of TB research, if researchers want to establish which human molecules are involved in different aspects of the disease process, they use a panel of fluorescently labelled antibodies that have been designed to target and bind to the molecules of interest. Different fluorescent dyes with different colours are used to label the antibodies. Sections of tissue are 'stuck' to microscope slides and the slides are flooded with a solution containing a panel of labelled antibodies. The slides are then washed, and only those antibodies that have bound to the molecules of interest remain, while antibodies that are not bound are washed off, together with their fluorescent labels.
By looking at the slides using a specialised microscope to visualise fluorescence, scientists can determine which molecules of interest are present and in what quantity. It this way, the light show can tell the story of what is happening between the antigens and antibodies.
Tracking gene expression
Another avenue of research involves looking at which of a person's genes are being expressed. Here too a little background aids understanding.
Enzymes are proteins that direct the metabolic processes in humans such as using or storing glucose, breaking down fats or producing antibodies. Humans have codes in their DNA (genes) that allow different cells to make thousands of different proteins. However, only a fraction of our genes is expressed or 'turned on' at any one time because we don't want to waste energy making proteins that are not needed. Transcriptomics is the study of the molecules (messenger RNA) involved in regulating the expression of genes.
Finding out which genes are 'turned on' in response to TB infection could provide valuable clues into how TB disease works. For example, Naidoo and the AHRI team are interested in which genes are expressed around granulomas - tiny clusters of cells and other tissue which are formed in reaction to infections or inflammation - in comparison to healthy tissues.
Finally, a technique called high-resolution proteomics can be used to look at the end products of gene expression, namely, the proteins themselves. More than 5 000 human proteins can be identified and compared in tiny pieces of infected and healthy tissues using laser capture microdissection (LCM) followed by mass spectrometry (MS). LCM is a technique used to isolate specific regions from a tissue sample under microscopic visualisation. Using a laser, researchers precisely cut and capture targeted cells or regions while leaving the surrounding tissue intact. The isolated material can then be used for MS analyses. MS is a analytical technique that allows for the detailed characterisation and identification of proteins in biological samples.
Where does all this fancy research technology get us?
In 2019, using high-resolution micro-computed tomography, scientists at AHRI made a discovery that advanced our understanding of what TB does in the body. Though not immediately relevant to people ill with TB, such 'basic research' forms the foundation on which diagnostic and treatment advances might be made down the line.
"Although we sectioned the tissue into very, very thin slices, five microns, on a slide ... and were looking at the round granuloma (tiny clusters of cells and other tissue which are formed in reaction to infections or inflammation) in the lung tissue, we wanted to see what a whole granuloma would look like, in its totality," says Steyn.
For decades, the necrotic granuloma has been widely assumed to have a round, ball-shape (spherical) and a lot of studies have been published on this assumption.
"However, we found that the granulomas generated by mycobacterium tuberculosis are not spherical as we thought previously from our 2D studies, but that they are shaped more like a 'ginger root' with many branches, and they follow the airways," says Steyn. "Our 3D visualisation of the granuloma - and the discovery that they are not shaped like spheres - was crucial as it can help us to understand how the infection affects the lung and spreads along the airways. For example, this finding highlights the likelihood that a single complex 'ginger root' could be erroneously viewed as multiple independent lesions when evaluated in 2D under the microscope. This is an important finding as it improves our understanding of how dissemination of mycobacterium tuberculosis in the lungs contributes to TB disease and provides a compelling rationale for considering aerosolised anti-TB drug delivery strategies."
In other words, the thinking is that given our new understanding of how TB spreads along the airways inside the lung, aerosolised TB treatments that people breathe in should be able to reach the bug via those same airways.
Steyn adds: "This discovery was a lightbulb moment for us. Arising from this, came the idea to establish a 3D atlas of the human tuberculosis lung. It's one thing to see a very thin section on a slide, but another to see a 3D atlas of the tuberculosis lung - and we are currently working on that and getting closer to achieving this goal."
Why tissue research?
Granulomas offer a good example of why tissue research is so important for our understanding of TB. As Naidoo explains: "The more accessible cells in the body would be blood, saliva and sputum, but studying them is looking at the effects of TB. If you want to study what TB is actually doing in the tissue, you need the tissue itself, as the primary site of infection."
And in the case of granulomas, looking at the tissue itself means looking at an actual granuloma. Since looking at a granuloma inside a living person's lung is difficult, not to mention dangerous, the most feasible way to actually get a look at it is through the type of tissue donations that AHRI do their research on.
The hope is that such a more direct look at what is happening in lung tissue will shed light on TB's remaining mysteries.
"TB has been particularly challenging because," says Naidoo, "while we have antibiotic therapy for it, the way it infects the body and the immune response it elicits, has allowed it to cause much more damage at tissue level and the treatment is not always completely effective. Even though a patient might be cleared of the organism, through a so-called microbiological cure, the damage caused by the organism and the body's immune response to that organism, needs to be understood better."
Mysteries of TB progression
Naidoo says one of the many things still not completely understood about TB is the exact mechanisms that cause the transition, from the bacteria being dormant or latent in the patient's lung, to causing active disease; how it can be contained naturally, sometimes for decades, without causing disease in most people, to then become active and uncontrolled.
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"There are different stages of the infection in humans, ranging from when the affected person is healthy and well to when they become sick and possibly suffer permanent complications after completing treatment. In order to conduct meaningful tissue research, you need to build a tissue repository that is representative of the full continuum of TB disease. You also need capacity to study it in its entirety, at the cellular and molecular level.," he says.
In addition to sourcing tissue from surgeries, the researchers also have an arrangement with local state mortuaries to get tissue samples from autopsies on people who died of causes other than TB, but who had TB in their lungs. Because these people died of causes other than TB, their tissue samples reflect various stages of TB infection. Spotlight previously reported on what we can learn about TB at the autopsy table here.
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Steyn goes into more detail to explain that they are trying to understand the precise process of how the TB organism causes disease and how it destroys tissue in the lung. "That process is completely unknown. We know more what's going on in a mouse lung than in human lungs. We need to understand the patho-physiological process, which means how the abnormalities causing the disease arise, how they progress to cause the disease, and why," says Steyn.
"Ultimately, our goal is to characterise all these changes and identify protein networks (proteins that work together) so that we can identify new drug targets (specific molecules, often a protein, in the body, that are linked to a particular disease and can be targeted by a drug). We would like to identify a drug target which is host directed, i.e., a host process that can be targeted to kill the bacterium as opposed to looking at drugs that kills the bacterium. We want to understand what's going on in the host - the human that is infected by the TB bacillus. We want to know what went wrong in the host. If we understand that, then we should be able to identify new anti-TB drugs.
Steyn says there is also the strong hope to identify and develop new innovative TB tests. "If we don't know how the pathogen caused disease, then it's very difficult to come up with new diagnostics. We have to understand what went wrong, so that we can develop a diagnostic kit (a set of tests to make a diagnosis). And then..., one cannot deny the fact that once you understand the disease process, you can't help thinking about the implications for vaccine design. Once we understand what went wrong, it will provide us with a much better foundation for developing new vaccines."
