Blood-brain-barrier spheroids present high level of efflux pump (left: green) and tight junctions (center and right: green) on the surface of each spheroid to keep foreign molecules out.

Building a Better Blood-Brain Barrier Model

Delivering drugs to the brain is no easy task. The blood-brain barrier –a protective sheath of tissue that shields the brain from harmful chemicals and invaders – cannot be penetrated by most therapeutics that are injected into a person’s blood stream. But for treating diseases of the central nervous system and cancers such as glioblastoma, it’s essential to get drugs across this barrier and deliver them to where they are needed most. Current research models that are used to study or imitate the blood-brain barrier have a number of limitations. Investigators at Brigham and Women’s Hospital have developed an innovative but easily implemented approach that uses “spheroids” to mimic the blood-brain barrier more accurately, and appears to overcome several challenges for discovering and advancing new drugs for treating brain conditions. They report their results June 6 in Nature Communications.

“Our model takes a new approach to mimic the blood-brain barrier outside of a living system. These miniature spheroids are relatively straightforward to culture, and yet it is able to reproduce many of the key blood-brain barrier properties and functions,” said lead author Choi-Fong Cho, PhD, of the Department of Neurosurgery. “Our hope is that these findings will further advance neuroscience research and expedite the discovery and design of brain-penetrant drugs to treat diseases of the brain and central nervous system.”

Current models of the blood-brain barrier rely largely on either animal models – which are expensive, laborious and can only be used to test a limited number of compounds at a time – or cells grown in the lab. For the latter, it’s very difficult to replicate the conditions found in the human body. Cells are often grown on flat plastic surfaces, isolated by cell type, and may become less and less like the unique cells found in the brain over time. The new technique grows different kinds of brain cells – endothelial cells, pericytes and astrocytes – together, allowing them to spontaneously form multicellular spheroids. These self-assembled structures closely resemble the blood-brain barrier organization and can be used to predict drug penetration capabilities – molecules that can penetrate the surface of the spheres and accumulate inside are more likely to be able to penetrate the blood-brain barrier and enter the brain in a living organism.

The research team performed several tests on these spheroids to establish some of the key properties of the blood-brain barrier that allow it to restrict the influx of foreign molecules. The spheroid model scored much better on many of these properties than the standard model in use today. The team also used the spheroids to identify new brain-penetrant molecules, which could hold high potential for delivering therapeutics across the blood-brain barrier.

“We plan to use this model going forward in our own research to identify new therapeutics for glioblastoma,” said senior author Sean Lawler, PhD, of the Department of Neurosurgery. “This is a very versatile model and should allow our group and others to test not only molecules but also viruses, cells and more that may be able to cross the blood-brain barrier.”

This work was supported by a Canadian Institute of Health Research Post-Doctoral Fellowship (1122374), a National Science Foundation Graduate Research Fellowship, a David H. Koch Graduate Fellowship Fund, a Sontag Distinguished Scientist Award and a National Cancer Institute grant (5-RO1-CA166172).

Paper cited: Cho CF et al. “Blood-brain-barrier spheroids as an in vitro screening platform for brain-penetrating agents” Nature Communications DOI: 10.1038/ncomms15623

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Using a microRNA to Shift the Makeup of Glioblastoma Subtypes

Glioblastoma multiforme (GBM), an extremely aggressive brain cancer, is a very complex disease. It is characterized by a fast-growing tumor in the brain composed of many subpopulations of cells, including glioblastoma stem cells, which play a crucial role in glioblastoma initiation, expansion and therapy-resistance. GBM’s diverse make up – termed heterogeneity – is of clinical importance because it is a key factor that leads to treatment failure, allowing the tumor to become resistant to treatment or for cancer to recur.

One way to identify different glioblastoma subtypes is by looking at the specific microRNA expressed in the patient derived GBM stem cells. In several types of cancer cells, including glioblastoma cells, microRNA expression isn’t regulated properly. In a new study published in Cell Reports, BWH researchers examined a specific microRNA, miR-128, to help identify glioblastoma subtypes and to determine if altering the microRNA’s presence in glioblastoma cells could change the tumor’s subtype.

“RNA is increasingly recognized as a snapshot of a cell at a given moment in time and therefore gives unique insight into the disease biology,” said lead author Arun Kumar Rooj, PhD, of the Department of Neurosurgery at BWH. “Understanding the dynamic spectrum of cells and their non-coding RNA signatures is critical for advancing therapeutic strategies that will be capable of overcoming the complexity of this disease.”

The researchers looked at miR-128 expression in diverse populations of glioblastoma cells. They identified the “proneural” subtype as having high levels of miR-128 compared to the mesenchymal tumors, which had significantly lower levels of this particular microRNA. Interestingly, they also found that if they raised or lowered the levels of miR-128, they could induce one subtype of tumor to transition into a new subtype.

“Mesenchymal glioblastoma is extremely aggressive, highly heterogeneous and has the poorest chance of survival for patients,” said corresponding author Agnieszka Bronisz, PhD, of the BWH Department of Neurosurgery. “By altering the level of miR-128 in both mesenchymal and proneural tumors, we can shift the tumor into a more hybrid type, similar to the “classical” subtype which is more homogenous and easier to treat.”

“The ability to transform more aggressive types of glioblastoma into a subtype that is more responsive to treatment opens the door for using miR-128 as a therapeutic agent,” said corresponding author Jakub Godlewski, PhD, of the BWH Department of Neurosurgery.

This work was funded by the National Cancer Institute (grant numbers P01 CA69246 and R01 CA 176203-01A1).

Paper cited: Rooj AK et al. “MicroRNA-Mediated Dynamic Bidirectional Shift between the Subclasses of Glioblastoma Stem-like Cells” Cell Reports DOI:10.1016/j.celrep.2017.05.040

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Socioeconomic Stress During Pregnancy Impacts Infant Brain Development

Children born into socioeconomically disadvantaged homes face higher risks of disease and health disorders throughout their lives. In fact, economic disparities can impact children’s future health before they are even born. The impact of social environment on a baby’s brain development is not fully understood, but new research led by investigators at BWH and the National Institute of Child Health and Development provides evidence that this connection is, in part, impacted by the mother’s immune response during pregnancy. Their findings are published this week in the Proceedings of the National Academy of Sciences.

Pregnancy can be a stressful time, especially for those under chronic financial and social strain. When an expectant mother is under chronic stress, her body’s immune system creates a physiological response that can influence fetal development. To understand how the mother’s immune system during pregnancy might influence neurodevelopment in a child’s early life, researchers examined the inflammatory response to chronic social adversity in 1,494 participants from the New England Family Study.

The researchers measured the concentrations of specific inflammatory chemicals, called cytokines, in maternal sera collected from women in their third trimester. They found that the most socioeconomically disadvantaged women had lower levels of interleukin-8 (IL-8), a pro-inflammatory cytokine. When stressed, the body produces “stress hormones” (glucocorticoids) that reduce the body’s inflammatory response. Chronic socioeconomic stress during pregnancy and the associated production of glucocorticoids may, in part, explain the decreased levels of IL-8.

This immune response can also impact a child’s brain development. In this study, decreased fetal exposure to IL-8 was significantly associated with neurologic abnormalities during infancy. Researchers found that children born to mothers with the largest socioeconomic disadvantage were 4.6 times more likely to have neurologic abnormalities at age 4 months and two times more likely to have these abnormalities at 1 year compared to the children of less disadvantaged mothers.

“The impact of social inequalities on health outcomes have early origins that may be retained throughout life,” said Jill Goldstein, PhD, Chair of the Brigham Research Institute Center for Research on Women’s Health and Gender Biology and senior author of the study. “Chronic socioeconomic stress during pregnancy, and the immune response that follows, can contribute to vulnerabilities later in life for offspring.”

However, Goldstein cautions that children born into poverty are not destined to experience more health problems. It is important to identify high risk children, which could result in intervening early to promote cognitive and emotional health.

This work was funded by the Office for Research on Women’s Health, the National Institute of Mental Health, the National Heart, Lung, and Blood Institute, and the Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development.

Paper cited: Gilman, S et al. “Socioeconomic disadvantage, gestational immune activity, and neurodevelopment in early childhood.” PNAS DOI: 10.1073/pnas.1617698114

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Understanding HIV’s Persistence

Most cells in the human body have a limited lifespan, typically dying after several days or weeks. And yet, HIV-1 infected cells manage to persist in the body for decades. Current treatment for HIV is very effective at suppressing the virus, but is unable to entirely clear the disease, which can rapidly recur if treatment is ever stopped. A new study, published in the Journal of Clinical Investigation, led by Mathias Lichterfeld, MD, PhD, and Guinevere Lee, PhD, from the BWH Infectious Disease Division sheds new light on the mechanism underlying the persistence of HIV-1 infected cells despite antiviral treatment.

“Our research points to a driving force that stabilizes the pool of HIV-infected cells in the host, which can persist lifelong despite very effective antiretroviral therapy,” said Lichterfeld. “These findings have important implications for efforts to reduce or eliminate HIV from the body, including interventions like vaccines and checkpoint inhibitors.”

Using a novel viral sequencing approach to track viral infection in different subtypes of CD4 T cells, this study found that a remarkable number of infected cells harbor sequences that are completely identical over the entire full-length viral sequence. Indeed, individual clusters of cells harboring such identical sequences were observed in roughly 60 percent of all memory CD4 T cells, the primary target cells for HIV. These data suggest that cells harboring identical viral sequences all stem from one particular CD4 T cell that presumably got infected prior to the beginning of antiviral therapy. That cell has gone on to disseminate and expand the HIV-infected cell pool whenever it divides, passing on the viral genetic material to its daughter cells in a process called “clonal proliferation.” By this mechanism, a single HIV-infected cell can, simply by dividing for 10-20 times, amplify the number of virally infected cells by up to a million fold.

“This work shows that HIV is taking a free ride: It effectively exploits the normal proliferative behavior of human cells for propagation and dissemination of the viral genome,” said Lee.

Interrupting or blocking proliferation of such virally-infected cells may represent a future strategy to limit viral persistence despite treatment, and may someday allow for the development of novel clinical interventions, leading to a long-term, drug-free remission of HIV infection.

Funding for this work was provided by the National Institutes of Health (grants AI098487, AI106468, AI114235, AI117841, AI120008, AI124776, AI116228, AI078799, HL134539, R3767073), the AIDS Vaccine Discovery from the Bill and Melinda Gates Foundation, the International AIDS Vaccine Initiative, the NIH-funded Harvard University Center for AIDS Research (grant P30 AI060354) which is supported by the following NIH co-funding and participating Institutes and Centers: NIAID, NCI, NICHD, NHLBI, NIDA, NIMH, NIA, FIC, and OAR.

Paper cited: Lee GQ et al. “Clonal expansion of genome-intact HIV-1 in functionally-polarized Th1 CD4 T cells.” Journal of Clinical Investigation DOI: 10.1172/JCI93289

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In the absence of claudin-2, clearance of C. rodentium (red) is impaired and bacteria are able to invade the normally-protected colonic crypt lumen. F-actin (green) and DNA (blue),

Why We Get Diarrhea

Researchers want to know: does diarrhea serve a purpose? Does it actually help clear the bacteria causing a gastrointestinal infection, or is it merely a symptom of disease that should be prevented as much as possible? In a new study from Brigham and Women’s Hospital, investigators explore the immune mechanism that drives diarrhea, concluding that it does play a critical role in pathogen clearance in the early stages of infection. The new study, published today in Cell Host and Microbe, also uncovers a previously unrecognized role for interleukin-22, an immune system molecule, in the host’s defense against infection.

“The hypothesis that diarrhea clears intestinal pathogens has been debated for centuries,” said corresponding author Jerrold Turner, MD, PhD, of the BWH Departments of Pathology and Medicine. “Its impact on the progression of intestinal infections remains poorly understood. We sought to define the role of diarrhea and to see if preventing it might actually delay pathogen clearance and prolong disease.”

To investigate, researchers used a mouse model infected with Citrobacter rodentium, the mouse equivalent of an E. coli infection. Using this model, they saw an increase in the permeability of the intestinal barrier within just two days of infection – well before inflammation and epithelial damage. In particular, they uncovered a critical role for interleukin-22 that in turn influences another molecule called claudin-2, previously known to be involved in causing diarrhea. They found that diarrhea resulting from the signaling of these two molecules helped promote pathogen clearance and limited disease severity.

Other investigators have proposed developing new therapeutics to inhibit claudin-2. However, Turner and colleagues explain that the activation of this pathway may be critical for combating an infection, particularly in the early stages of a disease. They conclude that diarrhea is critical to enteric pathogen clearance, and that IL-22 may play a key role in host defense.

This work was supported by National Institute of Health grants F30DK103511, T32HD007009, R01DK61931, R01DK68271, and R24DK099803; Crohn’s and Colitis Foundation of America; the State Scholarship Fund of China 201208110294; and Core Research for Evolutional Science and Technology of the Japan Science and Technology Agency.

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Paper cited: Tsai PY et al. “IL-22 upregulates epithelial claudin-2 to drive diarrhea and enteric pathogen clearance.” Cell Host & Microbe DOI: 10.1016/j.chom.2017.05.009

New Class of Type 2 Diabetes Drug Associated with Rare, Life-Threatening Outcome

A new class of drugs, known as SGLT2 inhibitors, is increasingly being prescribed for the treatment of type 2 diabetes, but may increase the risk of rare but serious complication known as diabetic ketoacidosis. In a new study published in The New England Journal of Medicine, investigators from Brigham and Women’s Hospital quantify that risk, finding that patients are twice as likely to experience diabetic ketoacidosis if taking an SGLT2 inhibitor rather than another class of diabetes drugs. However, diabetic ketoacidosis is still extremely rare: even for patients taking an SGLT2 inhibitor, only about one in every 1,000 patients will experience this complication, the researchers estimate.

The research team studied 40,000 patients taking SGLT2 inhibitors, comparing their outcomes to those of patients taking a DPP4 inhibitor. After 180 days, 55 patients taking an SGLT2 inhibitor had experienced diabetic ketoacidosis, while 26 patients taking the other class of drug had experienced this side effect.

SGLT2 inhibitors were first brought to market in April of 2013 and, based on clinical trials data, appeared to be quite safe. However, case reports of diabetic ketoacidosis among people with type 2 diabetes taking SGLT2 inhibitors prompted the FDA to issue a warning in 2015 about the class of drugs. Diabetic ketoacidosis is usually associated with type 1 diabetes – it’s very uncommon for people with type 2 diabetes to experience this complication. Those who do suffer from this complication have high levels of acids, called ketones, in their blood and can experience vomiting, abdominal pain, shortness of breath, swelling in the brain and, if left untreated, diabetic ketoacidosis can be fatal.

Corresponding author Michael Fralick, MD, FRCPC, of the BWH Division of Pharmacoepidemiology and Pharmacoeconomics, emphasizes that even though diabetic ketoacidosis is uncommon, physicians need to be vigilant for signs and symptoms among type 2 diabetes patients.

Fralick became interested in exploring the association between SGLT2 inhibitors and this side effect after one of his patients who had been taking this medication came to the emergency room with symptoms of diabetic ketoacidosis. Using data available to him through one of the research platforms available at the Division (Aetion Evidence Platform) he was able to conduct this study within weeks of seeing this patient.

“My best research projects come from my patients – their experiences drive the questions I investigate,” Fralick said. “This is a side effect that’s usually seen in patients with type 1 diabetes mellitus – not type 2 – so doctors are not ‘on the lookout’ for it. That means that the risk of this side effect might actually be even higher than what we found due to misdiagnosis/under recording.”

Support for this work was provided by the Division of Pharmacoepidemiology and Parmacoepidemiology.

Paper cited: Fralick M et al. “Risk of Diabetic Ketoacidosis after Initiation of an SGLT2 Inhibitor” New England Journal of Medicine DOI: 10.1056/NEJMc1701990

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