Immunotherapy—Don’t Forget the Microbiome

Bacteria make you sick. The idea that bacteria cause illness has become ingrained in modern society, made evident by every sign requiring employees to wash their hands before leaving a restroom and the frequent food recalls resulting from pathogens like E. coli. But a parallel idea has also taken hold. As microbiome research continues to reveal the important role that bacteria play in human health, we’re starting to see the ways that the microbiota of the human body may be as important as our genes or environment.

The story of how our microbiome affects our health continues to get more complex. For example, researchers are now beginning to understand that the composition of bacteria residing in your body can significantly impact the effects of therapeutic drugs. This is a new factor for optimizing drug response, compared to other considerations such as diet, interaction with other drugs, administration time and comorbidity, which have been understood much longer.

The real complexity arises in the myriad ways that bacteria can affect a drug. For example, some bacteria may produce an enzyme that converts a drug compound into a new molecule, often resulting in a reduction of potency. The new molecule can be chemically inactive or no longer absorbed by the target tissue.

Interactions between bacteria and the immune system can pose special challenges for researchers and doctors employing immunotherapy to treat cancer, which can have response rates as low as 12%. The microbiome may be to blame in many cases. For example, anti-PD1 immunotherapy yielded very different response rates in patients with melanoma depending on the characteristics of their microbiome. Patients with certain species present in their gut had better response rates than those without them.

The question that remains is how the bacteria are influencing the drug action of immunotherapies. In addition to producing molecules that act directly on drug compounds, there are a number of other mechanisms through which bacteria can impact an individual’s response to immunotherapy.

Bacteria could play a role in training or priming the immune system. If certain bacterial antigens are similar to tumor antigens, the immune system can gain the molecular tools and memory needed to fight cancer. Individuals without such bacteria would have an immune system naïve to the cancer antigens, giving a tumor more time to fly under the radar and gain mutations that further evade the immune system.

Response rates to immunotherapy in mice have been shown to decrease if treatment was preceded by a course of antibiotics. When the mouse microbiome was disrupted, the number of immune cells declined. This resulted in a decline in tumor necrosis factor (TNF) production. TNF is needed to enable CpG-oligodeoxynucleotides, the type of immunotherapy used in this study, to induce tumor necrosis. Mice that weren’t treated with antibiotics had a greater reduction in tumor size from the treatment. Left undisturbed, their microbiome appeared to prime the immune cells to secrete TNF.

In another mouse study, treatment with tumor specific CD8+ T cells was more likely to work if the mice were irradiated first. The radiation irritates the lining of the gut, freeing bacteria to travel to other parts of the body. Once the microbes establish themselves in a destination, they produce lipopolysaccharides that stimulate immune cells in the vicinity. The researchers also found that gut microbes promote dendritic cell maturation which activate CD8+ T cells and induce them to kill tumor cells.

You may be thinking these examples just reveal a few drugs that are affected by the microbiome, but one study shows that bacterial influence on drug action is probably the rule rather than the exception. After testing nearly 300 drug compounds, one group of researchers found that two-thirds were modified by bacteria in some way. The fact of the matter is that bacteria are responsible for most of the molecules found in our bloodstream, which must be taken into account when measuring drug efficacy.

The key to boosting the success rate for immunotherapy and drug treatments for cancer and many other diseases probably lies in a cotreatment targeting bacteria in the gut. Probiotics, prebiotics, fecal transplants and even fiber-rich diets are all being examined in clinical studies to determine if they should become part of standard treatment alongside drug therapies for a variety of diseases.

Studies like these expose the conceit of humans trying to control disease. Although the paradigm was once to destroy bacteria and sterilize the environment to prevent and treat the diseases they cause, there is now a shift toward nurturing and manipulating bacteria to provide an optimal environment for therapeutic compounds. While precision medicine entered the stage by championing the subtle genetic differences between individuals as the key to curing cancer, it may be the genes of the organisms we harbor that prove to be a more significant factor.

Learn more about microbiome research:

Feature Article: Metagenomics, Microbes and the Meaning of Life: From subways to space stations and beyond

Webinar: Metagenomic Mapping of Medical, Urban and Space Environments

Related Posts

Kinase Inhibitors as Therapeutics: A Review

This blog was originally published in April of 2018. Today’s update includes the paper, “Quantitative, Wide-Spectrum Kinase Profiling in Live Cells for Assessing the Effect of Cellular ATP on Target Engagement” from Cell Chemical Biology, demonstrating the power of NanoBRET™ target engagement kinase assays in the study of kinase inhibitors.

The review “Kinase Inhibitors: the road ahead” was recently published in Nature Reviews Drug Discovery. In it, authors Fleur Ferguson and Nathanael Gray provide an up-to-date look at the “biological processes and disease areas that kinase-targeting small molecules are being developed against”. They note the related challenges and the strategies and technologies being used to efficiently generate highly-optimized kinase inhibitors.

This review describes the state of the art for kinase inhibitor therapeutics. To understand why kinase inhibitors are so important in the development of cancer (and other) therapeutics research, let’s start with the role of kinases in cellular physiology.

The road ahead for kinase inhibitor studies.

Why Kinases? Continue reading “Kinase Inhibitors as Therapeutics: A Review”

Brazilian University Swatting at Leishmaniasis Parasite

The Medicinal Chemistry Center (CQMED), headquartered at Campinas State University in Brazil, recently started a project in partnership with Promega to develop drugs that can be used against Leishmania. This genus of protozoans is the etiological agent of leishmaniasis, transmitted to humans by sandflies.

Microscopic image of Leishmania parasite
Microscopic image of Leishmania tropica. Credit: Brian E. Keas at Michigan State University.

Leishmaniasis is classified as a neglected tropical disease that mainly affects poor communities. Symptoms include large skin sores and an enlarged spleen. The challenge in developing drugs to treat Leishmania is finding appropriate therapeutic targets. These targets are normally proteins whose inhibition leads to death of the parasite. In addition to pharmaceutical company Eurofarma, whose goal is to develop drugs for Leishmania, Promega was chosen to help solve this problem because of our NanoBRET™ Target Engagement (TE) assay*, a well-established technique for measuring protein interactions. In this assay, NanoLuc® luciferase is attached to the protein of interest, and a fluorescent NanoBRET™ tracer molecule is added to the cells. This produces a BRET signal. When a competing ligand is added, it will displace the tracer molecule, enabling quantification of the strength of the interaction compared to the tracer molecule..

A challenge that researchers will face will be ensuring that the NanoBRET™ tracer reaches the inside of the parasite cells; because Leishmania is an intracellular parasite, molecules need to cross the host cell membrane, the membrane of the vacuole containing the parasites, and the membrane of the parasite itself. Another challenge the slow reproduction of Leishmania within macrophages. On top of that is the fact that the parasite’s metabolism varies depending on its biological cycle, meaning that there could be long periods of time during which a drug’s therapeutic target is not expressed in the cell, during which time the drug would have no effect. The ideal target would be expressed at high levels throughout the cell cycle.

The project is being led by Rafael Couñago, a researcher at CQMED, and Promega scientists Matt Robers and Jean-Luc Vaillaud.

*An earlier version of this blog incorrectly said that these experiments are based on the NanoBRET™ assay using HaloTag® protein.

“The Human Placenta,” or “Why I Love Science Writing”

Have you read last week’s breaking story about the microbiome of the human placenta? Wait, stop, don’t run away to Google it! I’ll tell you all about it – this is a science blog, remember?

I’m asking because as I started reading about the topic in preparation for writing this blog post, I noticed two things. First, as a science writer who tries to stay well-connected with what’s going on in the world of biology research, it would have been nearly impossible for me to avoid this story. I get eight or nine daily digest emails from scientific publications every day, and I think over the course of last week, every single one came with a headline related to the placenta study. (Of course, I read them all. And the Nature study they were based on.)

Second, I noticed that each story I read had a slightly different angle on covering the research. As scientists, we like to believe that science is, well, just science. It’s factual. We pore over the data and reach a conclusion. If we aren’t sure of something, we search the journals. The story, if there is one, is about methods and controls, protocols and reagent quality. However, when information about that research is communicated broadly, outside of the journals, we can get a different impression based on how the author frames their article. Continue reading ““The Human Placenta,” or “Why I Love Science Writing””

qPCR: The Very Basics

Real-Time (or quantitative, qPCR) monitors PCR amplification as it happens and allows you to measure starting material in your reaction.
Real-Time (or quantitative, qPCR) monitors PCR amplification as it happens and allows you to measure starting material in your reaction. Data are presented graphically rather than as bands on a gel.

For those of us well versed in traditional, end-point PCR, wrapping our minds and methods around real-time or quantitative (qPCR) can be challenging. Here at Promega Connections, we are beginning a series of blogs designed to explain how qPCR works, things to consider when setting up and performing qPCR experiments, and what to look for in your results.

First, to get our bearings, let’s contrast traditional end-point PCR with qPCR.

End-Point PCR qPCR
Visualizes by agarose gel the amplified product AFTER it is produced (the end-point) Visualizes amplification as it happens (in real time) via a detection instrument
Does not precisely measure the starting DNA or RNA Allows you to measure how many copies of DNA or RNA you started with (quantitative = qPCR)
Less expensive; no special instruments required More expensive; requires special instrumentation
Basic molecular biology technique Requires slightly more technical prowess

 

Quantitative PCR (qPCR) can be used to answer the same experimental questions as traditional end-point PCR:  Detecting polymorphisms in DNA, amplifying low-abundance sequences for cloning or analysis, pathogen detection and others. However, the ability to observe amplification in real time and detect the number of copies in the starting material allow quantitation of gene expression, measurement of DNA damage, and quantitation of viral load in a sample and other applications.

Anytime that you are preforming a reaction where something is copied over and over in an exponential fashion—contaminants are just as likely to be copied as the desired input. Quantitative PCR is subject to the same contamination concerns as end-point PCR, but those concerns are magnified because the technique is so sensitive. Avoiding contamination is paramount for generating qPCR results that you can trust.

  1. Use aersol-resistant pipette tips, and have designated pipettors and tips for pre- and post-amplification steps.
  2. Wear gloves. Change them frequently.
  3. Have designated areas for pre- and post-amplification work.
  4. Use “master mixes” to minimize variability. A master mix is a ready-to-use mixture of your reaction components (excluding primers and sample) that you create for multiple reactions—because you are pipetting larger volumes to make it, and all of your reactions are getting their components from the same master mix, you are reducing variability from reaction to reaction.
  5. Dispense your primers into aliquots to minimize freeze-thaw cycles and the opportunity to introduce contaminants into a primer stock.

These are very basic tips that are common to both end-point and qPCR, but if you get these right, you are off to a good start no matter what your experimental goals are.

If you are looking for more information regarding qPCR, watch this supplementary video below.

 

Optimizing PCR: One Scientist’s Not So Fond Memories

primer_tubesThe first time I performed PCR was in 1992. I was finishing my Bachelors in Genetics and had an independent study project in a population genetics laboratory. My task was to try using a new technique, RAPD PCR, to distinguish clonal populations of the sea anemone, Metridium senile. These creatures can reproduce both sexually and asexually, which can make population genetics studies challenging. My professor was looking for a relatively simple method to identify individuals who were genetically identical (i.e., potential clones).

PCR was still in its infancy. No one in my lab had ever tried it before, and the department had one thermal cycler, which was located in a building across the street. We had a paper describing RAPD PCR for population work, so we ordered primers and Taq DNA polymerase and set about grinding up bits of frozen sea anemone to isolate the DNA. [The grinding process had to be done using a mortar and pestle seated in a bath of liquid nitrogen because the tissue had to remain frozen. If it thawed it became a disgusting mass of goo that was useless—but that is a topic for a different blog.] Since I had never done any of the procedures before, my professor and I assembled the first set of reactions together. When we ran our results on a gel, we had all sorts of bands—just what he was hoping to see. Unfortunately, we realized that we had added 10X more Taq DNA polymerase than we should have used. I repeated the amplification with the correct amount of Taq polymerase, and I saw nothing. Continue reading “Optimizing PCR: One Scientist’s Not So Fond Memories”

When Five Hundred Tigers Are Not Enough—Ten Years Later

Ten years ago, I wrote about the distressing news of lack of genetic diversity in the wild Amur tiger population. International Tiger Day seemed like a good time to check in on what progress has been made to both sustain and establish wild tiger populations worldwide. In 2010, 13 tiger range countries (TRC) committed to a goal of doubling the world’s tiger population by 2022.

Amur Tiger.

That timeline was an ambitious goal, as highlighted by a report published in PLOS One in November of 2018 (1). The authors assessed the recovery potential of 18 sites identified under the World Wide Fund for Nature’s (WWF) Tigers alive initiative. The recovery system has several parts: A source site with higher density of tigers that the area around it and has a legal framework that does (or will) protect the tiger population; a recovery site that has a lower density of tigers than the surrounding regions, has the ability to support more tigers but is not as supported as a source site; and a support region that connects a source and recovery site. These different site types all require different levels of management, available resources and legal protections, but they need to be managed in a coordinated way.

Aside from what is needed to manage these recovery sites, there are also other things that need to exist to support recovery of tiger populations. Some of these include support from local populations and governments, as well as environmental requirements such as breeding habitats and prey populations. For 15 of the 18 sites it is the prey population that is the sticking point. Recovery of prey populations is a slow process. The authors concluded that there need to be a commitment to achieving a realistic recovery of tiger populations, even if we miss the 2022 goal.

The fate of the wild tiger is still tenuous. Only time will tell if the interventions that are being implemented can be realized in time.

Reference

  1. Abishek, H. et al. (2018) Recovery planning towards doubling wild tigers Panthera tigris numbers: Detailing 18 recovery sites from across the range. PLOS One 13. e0207114. published online

Think Restriction Enzymes are so last decade? Not so fast!

Ribbon diagram of EcoRI homodimer bound to doublestranded DNA
Ribbon diagram of EcoRI homodimer bound to doublestranded DNA

Restriction enzymes sometimes get a lot of flak. In the not-so-distant past, they were the workhorses of molecular biology. Restriction enzymes played a huge role in developing early DNA sequencing techniques. They chop DNA in a predictable manner, which makes cutting and pasting genes of interest manageable and relatively easy, enabling the development of  genetic engineering and recombination technologies. These technologies are now moving beyond restriction enzymes toward more modern methods, with the most talked-about method being CRISPR /Cas9. As technology continues to advance at such a rapid pace, restriction analysis  and other “ancient” technologies feel antiquated. But this is not necessarily the case. Continue reading “Think Restriction Enzymes are so last decade? Not so fast!”

CRISPR/Cas9, NanoBRET and GPCRs: A Bright Future for Drug Discovery

GPCRs

G protein-coupled receptors (GPCRs) are a large family of receptors that traverse the cell membrane seven times. Functionally, GPCRs are extremely diverse, yet they contain highly conserved structural regions. GPCRs respond to a variety of signals, from small molecules to peptides and large proteins. Many GPCRs are involved in disease pathways and, not surprisingly, they present attractive targets for both small-molecule and biologic drugs.

In response to a signal, GPCRs undergo a conformational change, triggering an interaction with a G protein—a specialized protein that binds GDP in its inactive state or GTP when activated. Typically, the GPCR exchanges the G protein-bound GDP molecule for a GTP molecule, causing the activated G protein to dissociate into two subunits that remain anchored to the cell membrane. These subunits relay the signal to various other proteins that interact with or produce second-messenger molecules. Activation of a single G protein can result, ultimately, in the generation of thousands of second messengers.

Given the complexity of GPCR signaling pathways and their importance to human health, a considerable amount of research has been devoted to GPCR interactions, both with specific ligands and G proteins. Continue reading “CRISPR/Cas9, NanoBRET and GPCRs: A Bright Future for Drug Discovery”

Empowering Communities with the Light of the Sun

Today’s blog brought to you by Julia Nepper, a Promega science writer guest blogging for the BioPharmaceutical Technology Center Institute (BTC Institute)!

“We all benefit from STEM role models. When students from underrepresented populations meet and learn about STEM professionals of color, they can see themselves as the scientists and engineers of the future. Fun, engaging science programming for children is also essential to light the spark for the next generation. A Celebration of Life, the partnership between the BTC Institute and the African American Ethnic Academy, two community nonprofits, has combined these 2 objectives for over twenty years.” according to Barbara Bielec, K-12 Program Director.

This year, the theme of the program is Sunsational!, with a number of activities related to the sun, solar energy, and STEM careers. As part of the program, students heard talks from several STEM professionals of color about their work. Mehrdad Arjmand, co-founder of solar energy company NovoMoto, was one of those speakers.

Dr. Arjmand was born and raised in Iran. His path to becoming a mechanical engineer began as a child, with him “destroying a lot of equipment” in his house. After completing his undergraduate education, he came to the States to pursue a PhD at the University of Wisconsin-Madison, where he met Aaron Olson, a student who was born in the Democratic Republic of Congo. These two discovered a shared passion for starting a business and helping their communities, which led directly to the founding of NovoMoto. The name derives from Portuguese for “new” (novo) and Lingala—a language spoken in Congo—for “fire” (moto). Continue reading “Empowering Communities with the Light of the Sun”