#chlaorgdiscoveries

A new study, published online in European Respiratory Journal, marks an important milestone. “This is one of the very first studies looking at how the human lung develops at the single cell level,” says Dr. Al Alam. The study tracked the fate of these cells over time, showing a trajectory of cell development. Knowing when certain cell types are differentiating gives investigators a very detailed, time-lapse map to lung development.

Dr. Al Alam’s team focused on the development of two types of cells in the lung. Airway smooth muscle cells line structures such as the trachea, bronchial tubes, and smaller branches. Vascular smooth muscle cells are found in the walls of blood vessels. Both types of cells add muscle tone and stability while also mediating necessary movement within these structures. But they are often implicated in different disease pathways.

“The problem is that we can never really study one cell type without also studying the other,” says Dr. Al Alam, investigator in the Developmental Biology and Regenerative Medicine Program at The Saban Research Institute of CHLA. Up until now, investigators have used a marker – known as ACTA2 – to identify these cells. But the marker doesn’t distinguish between the two smooth muscle cell types. Although both types of cells are smooth muscle they are implicated in totally different pulmonary diseases. “We need to study the two types of cells independently to understand what happens in different lung disorders.” Fortunately, her work has solved this problem that has hindered research in the field.

Dr. Al Alam’s team has identified molecular markers that are unique to each subset of cells. This will allow investigators to differentiate between the two groups, which will benefit respiratory research efforts worldwide. Developmental disorders such as COPD, asthma, and bronchopulmonary dysplasia can now be studied separately from related, but distinct, conditions that arise later in life, such as pulmonary hypertension.

“It’s been decades since we’ve introduced anything into the clinic to treat lung diseases associated with prematurity,” says Dr. Al Alam. “We are putting together critical pieces so we can get these babies the treatments they need.”

In a landmark study published in Nature Communications, scientists at Children’s Hospital Los Angeles demonstrate an in vitro kidney model that could change the course of research for diseases like CKD.

The kidney contains specialized structures called glomeruli. Within each glomerulus is a filtration barrier made up of two thin layers of highly specialized cells and a membrane that acts as a selective filter. As blood moves through each glomerulus, toxins and small molecules can pass through, while proteins and other important components are kept in the bloodstream. “This filtration process breaks down in patients with kidney disorders,” explains Laura Perin, PhD, who is co-senior author on the study along with Stefano Da Sacco, PhD. “But because we haven’t had a good in vitro model, we still don’t know the mechanisms of injury to the glomerulus in CKD.”

Dr. Perin and Dr. Da Sacco conduct research in the GOFARR Laboratory for Organ Regenerative Research and Cell Therapeutics in Urology along with co-director Roger De Filippo, MD, at CHLA’s Saban Research Institute. The lead author on the study was CHLA postdoctoral research fellow Astgik Petrosyan. Together, the team studies the structure of the glomerulus to better understand how and why their ability to filter blood breaks down.

“A big challenge in the kidney research field has been trying to replicate the glomerulus in vitro,” says Dr. Da Sacco. “In particular, the glomerular filtration barrier is very difficult to recreate in a lab using standard techniques.” Because of this, most published studies have used an artificial membrane between the two cell layers. While fluid can be exchanged, the cells cannot communicate across this membrane in the same way they do biologically. “This results in a model that doesn’t really filter properly,” he explains.

The critical component missing from current experiments is a filter that is selective and allows proper cell-to-cell communication. Dr. Da Sacco and Dr. Perin set out to grow healthy kidney cells in a way that allowed for the natural glomerular barrier to form, just as it does in the body. Using specialized, compartmented containers called OrganoplatesTM, the investigators did exactly that.

The result?

A model glomerulus that functions nearly identically to that found in real kidneys. They are calling this model, which is derived entirely from healthy, human kidney tissue, a glomerulus on a chip.

On one side of the cells, investigators add fluid and, on the other side, they collect what the ‘glomerulus’ filters, which is called the filtrate. In their experiment, the scientists added blood serum from healthy individuals. Without the use of a manufactured filter, the team’s in vitro glomerulus behaved as human kidneys are expected to act: proteins remained in the serum while smaller molecules passed into the filtrate. “The barrier that our cells naturally formed is selective, just as it would be in a fully-functioning kidney,” says Dr. Da Sacco. “It is remarkable.”

This model represents a substantial leap forward from the current standard of in vitro kidney research. “Our system behaves like a biologically, physiologically correct glomerulus,” says Dr. Perin. “This opens up the door for us to understand what we still don’t know – the molecular mechanisms of injury in CKD and, more importantly, how to prevent damage.”

While this seemed a distant goal in the past, Dr. Da Sacco and Dr. Perin are already recreating and studying the disease state in their model. When the investigators added serum from patients with CKD, they found that the glomerulus exhibited the same type of damage observed clinically: proteins began to leak through the compromised filter. Protein levels measured in the experimental filtrate matched patient clinical filtrate samples with a correlation of approximately 90%.

This breakthrough paves the way for numerous clinical applications. In the burgeoning era of personalized medicine, a preparation such as this can be used to examine molecular mechanisms of kidney damage in individual patients. Disease progression can also be monitored over time using serial blood sampling. Also, the model could be used for screening new drugs prior to human clinical testing.

Dr. Perin and Dr. Da Sacco, who are also Assistant Professors in the Keck School of Medicine at USC, are co-senior authors on the publication. Other authors are Paolo Cravedi of Icahn School of Medicine at Mount Sinai, NY; Valentina Villani of CHLA; Andrea Angeletti of the University of Bologna, Italy; Joaquin Manrique of Complejo Hospitalario de Navarra, Pamplona, Spain; Alessandra Renieri of Azienda Ospedaliera Universitaria Senese, Siena, Italy; and Roger De Filippo of CHLA.

The study was funded by the GOFARR Foundation and the Schenkman Family, grants from the Alport Syndrome Foundation, a TSRI Research Career Development Award, and a Wright Foundation Pilot Award. OrganoplatesTM were purchased from MIMETAS, who the authors wish to thank for providing invaluable training, technical support, and assistance.

Healthy human kidney cells (visible in red and green in this fluorescent image) grow in layers to form a filtration barrier in vitro. Image courtesy of Dr. Perin and Dr. Da Sacco.

Whereas once this might have seemed like an out-of-reach goal, investigators and clinicians at CHLA are already implementing strategies to make this a reality. Patients are now able to benefit from CHLA’s OncoKids®, a specialized assay that tests a patient’s cancer for various biological markers. Results from the OncoKids panel can help doctors diagnose and treat pediatric patients based on the genetic makeup of their tumor. 

Earlier this week, the FDA granted accelerated approval for Vitrakvi® (larotrectinib), a drug that targets tumors with something called a TRK fusion – a fusion of two normal genes that, when combined, cause cancer. TRK fusions are estimated to cause up to 1% of solid tumors.  But unlike some cancer-causing mutations – like the BRCA mutation in breast cancer – TRK fusions can cause cancer in any tissue type in the body. Larotrectinib is an oral medication that targets TRK fusion. It is only the second drug approved by the FDA that is “tissue-agnostic,” not specific to the location of the tumor but instead targets tumors based on genetics. Development and approval of larotrectinib marks a move towards more targeted therapies for the treatment of cancer.

Mascarenhas oversaw the treatment of several patients at CHLA, including young Michelle, as part of the clinical trial for larotrectinib. Michelle’s story unfolded over the course of just a few days, when a tumor the size of a pea grew so large that it nearly crushed her airway. Through the use of the OncoKids® panel, doctors learned that Michelle’s cancer bore a TRK fusion.  In what can only be described as a well-orchestrated push to fight for Michelle, surgeons, oncologists, clinical research staff, and even pharmacists at CHLA worked around the clock to sign Michelle up for the clinical trial and get access to the medication.  This massive effort was not in vain – like the majority of patients involved in the trial, Michelle experienced a rapid, dramatic reduction in her tumor size.

This year, Mascarenhas co-authored articles in the New England Journal of Medicine and the journals Lancet Oncology and Cancer demonstrating results from the clinical trial. These reports describe larotrectinib’s “rapid, potent, and durable antitumor activity” in patients of all ages with TRK fusion tumors. Michelle can’t yet read these reports, but the result she likely cares most about was getting to return home to her family.

To the impressive feat of rapid reduction in tumor size (several had completely vanished), Mascarenhas adds that the side effects of the drug are minimal, “definitely nothing as severe as the side effects of chemotherapy,” Mascarenhas says.  Indeed, he describes larotrectinib as a “magic bullet” for tumors bearing TRK fusion.  “This drug doesn’t work in a particular type of cancer,” Mascarenhas says, “it works in any cancer that has the TRK fusion.  It works in lung cancer, colon cancer, salivary gland tumors, sarcomas, brain tumors, and thyroid cancers.  So that’s unique.”

How do striking results like these affect doctors who have dedicated their careers to caring for children with cancer?  “We were all amazed,” reflects Mascarenhas. “To see someone turn around from their death bed – it’s truly remarkable.”

Sebastien Bouret, PhD of the Saban Research Institute at CHLA leads a team of researchers that study the formation of these circuits. “We know that the brain, in particular an area called the hypothalamus, has a very important role in the regulation of food intake and blood sugar,” explains Dr. Bouret, who is also an associate professor of pediatrics at the Keck School of Medicine of USC. Researchers have focused on the hypothalamus for years in an effort to study the epidemic of obesity, which affects nearly 14 million children and adolescents in the United States. “What we don’t yet understand,” he says, “is how these circuits in the hypothalamus are being organized. We want to know how the brain puts itself together and what exactly governs that process.” Understanding this is key because circuits must be established properly in order for the brain to ultimately perform complex functions like maintaining proper weight.

Enter the semaphorins – small signaling molecules found in abundance in the developing hypothalamus. Bouret wondered whether these semaphorins could help form body weight circuits. Dr. Sophie Croizier, who led the study in Dr. Bouret’s lab, blocked semaphorin signaling in cells of the hypothalamus. She discovered that brain cells no longer grew the way they were supposed to, showing that semaphorin provides an essential map for them to follow. In addition to connections failing to establish, loss of semaphorin action in a preclinical model also caused elevated body weight. “What we are seeing is that semaphorins are guiding and shaping development of hypothalamic circuits that ultimately regulate calorie intake,” explains Dr. Bouret.

But the story does not stop here.

Professor Sadaf Farooqi, PhD, FRCP, FMedSci from the University of Cambridge was also analyzing genetic information from individuals with obesity. Dr. Farooqi’s team tested 1,000 DNA samples and found that individuals with early-onset obesity harbored more rare mutations in genes involved in semaphorin signaling than healthy individuals. The finding that people with obesity have these mutations shows that semaphorins are important in maintaining healthy body weight. “We have now discovered the genes that establish the precise neural connections that form these circuits,” says Dr. Agatha van der Klaauw, who led the study in Dr. Farooqi’s lab and is co-first author on the paper.

Many infants born premature or with other complications often forego their first weeks or months at home for a stay in the neonatal intensive care unit. The NICCU is designed to deliver critical medical care to babies in need but can be traumatic for infants and their families, alike. In the Early Childhood Mental Health Program at Children’s Hospital Los Angeles, clinical psychologists Marian Williams, PhD, Patricia Lakatos, PhD, and a team of infant-family mental health specialists work towards greater mental health awareness in the NICCU.

Infants may not be the first age group called to mind in discussions of mental health. Yet, for babies in critical medical condition, Dr. Lakatos says an “infant mental health-informed perspective” could reduce stress and improve bonding with parents. This means not only focusing on the physical needs of the child but also the emotional and mental needs, not an easy task for newborn infants who cannot make their voices heard.

In an article published in Journal of Clinical Psychology in Medical Settings, Dr. Lakatos, Dr. Williams, and co-authors Tamara Matic, MD, and Melissa Carson, MD, advocate for a third component of the NICCU family – the relationship between baby and parents. “A lot of mental health work in NICCUs currently focus on either the mental health of parents or on the baby’s development,” says Dr. Williams, who is also the Director of the Stein Tikun Olam Infant-Family Mental Health Initiative at CHLA. “We also want to focus on the relationship between babies and their parents.”

Many parents of children in intensive care units experience symptoms of post-traumatic stress, which can threaten bonding with a newborn baby. In order to support the developing relationship between parents and their new baby, the CHLA infant mental health team turned to a model of intervention that has demonstrated success in families who have undergone trauma. Child-Parent Psychotherapy – or CPP – addresses the parent-child relationship directly, nurturing and advocating for it in its own right.

With funding from the Stein Tikun Olam Infant-Family Health Initiative, Drs. Williams and Lakatos, and the team were able to adapt CPP to the NICCU setting at Children’s Hospital Los Angeles. Their publication describes how the established, evidence-based CPP model can be used to nurture developing infant-parent relationships in the NICCU. While it has been implemented in other settings, CPP is not commonly integrated into NICCU patient care.

CPP is a flexible model that has multiple levels of intervention, depending upon individual family needs. Sessions with trained CPP providers can vary in number or duration, with the aim of restoring a developmental trajectory for parent and child. CPP providers advocate for mental health needs of parents and babies, working alongside their medical and social work colleagues. “When babies are in the hospital, we need to think about them, their parents, and their relationships,” says Dr. Lakatos.