Delivering Drugs with Sound, Part 2: Treating muscles to treat the brain

Written by: Ken Estrellas

Original Article: Li et al. Molecular Therapy Methods & Clinical Development 2020
The Gist of It
You may recall that in my last post, I presented a study that used ultrasound waves, high-frequency sound waves typically undetectable by human hearing, to deliver drugs to cartilage. A recent study published by Li and colleagues extended this concept to a different area of the body – the brain – and applied it to a significant clinical issue that my fellow contributor Abel Cortinas wrote about in the final post of 2019: Alzheimer’s disease (AD). As Abel mentioned, a protein called amyloid beta (Aβ) clumps up in the brain of patients with AD, forming plaques associated with the disease. Previous studies have shown that there is less of an enzyme called neprilysin in the brains of patients with AD; also, getting rid of neprilysin increases the amount of Aβ made. Consequently, increasing the amount of neprilysin might help reduce the presence of these clumps, potentially relieving the symptoms of AD. However, increasing levels of neprilysin in the brain can be difficult from a practical standpoint, since administering the enzyme itself to the brain or using viral gene therapy to produce the enzyme both come with a significant risk of complications. In this study, the authors take an interesting approach: using ultrasound to inject modified genetic material known as plasmids that can lead to the production of neprilysin. However, instead of injecting this gene therapy directly into the brain, the leg was selected as an injection site instead. Since muscles contain a large number of blood vessels, the injected plasmids are able to circulate throughout the body, including the brain. The authors tested this approach in mice with a form of AD and observed that mice treated with ultrasound injections of plasmid had both more neprilysin and less Aβ protein in the brain one month after injection. These treated mice were also better able to navigate a complex water maze than untreated AD mice, suggesting that cognitive decline (a symptom of AD) had slowed. Importantly, the treated mice showed no signs of damage to the injected muscles or systemic damage in the kidneys. Overall, this study presents an alternative approach to the treatment of AD that appears promising and deserves further investigation.
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Ultrasound was used to deliver a gene therapy to muscle, with the effects observed in the brain

The Nitty Gritty
In this study, Li et al. explored the potential of ultrasound-assisted administration of 11,090 base pair plasmids to hindlimb muscles to more effectively overexpress neprilysin in the brain tissue of APP/PS1 mice, a model of AD, decreasing local concentrations of Aβ protein. The plasmids, which mediated overexpression of human neprilysin (hNEP) under the control of a cytomegalovirus (CMV) promoter, were delivered via microbubbles into the gastrocnemius muscles of N = 20 APP/PS1 mice with the simultaneous administration of 1.7 MHz, 1.0 W/cm2 ultrasound irradiation for one minute. One month after injection, increased levels of neprilysin were observed in the brains and skeletal muscles of hNEP-treated APP/PS1 mice compared with levels in untreated APP/PS1 mice, as determined by western blot. Significantly decreased levels of Aβ were detected in the brains of treated mice via western blot after one month compared with the levels in untreated mice, and this was visually observed via immunohistochemistry. A Morris Water Maze was used to compare potential functional improvements in treated vs untreated mice; treated mice showed a significantly decreased time to find a hidden platform in the shallow water maze (latency) and traveled a significantly shorter distance to do so (distance) after one month of treatment, whereas wild type C57/BL control mice and untreated APP/PS1 mice did not demonstrate any differences. Histological assessment of skeletal muscle, lung, and kidney tissue from treated mice using hematoxylin and eosin (H&E) staining revealed no signs of significant damage after one month. These results appear to present an initially promising strategy to overexpress neprilysin specifically in brain tissue without the need to cross the blood-brain barrier with recombinant enzyme or the complications associated with virally mediated gene therapy. Nonetheless, the authors acknowledge the need to assess the half-life of overexpressed hNEP, and the potential for long-term functional improvements – or declines – remain unknown.
Original Research Article: Li, Y., et al. “Expression of neprilysin in skeletal muscle by ultrasound-mediated gene transfer (sonoporation) reduces amyloid burden for Alzheimer’s disease.” Mol Ther Methods Clin Dev (2020) [Epub ahead of print].

Malaria: When fighting infection in your body doesn’t actually fight infection for the community

Written By: Rebecca Tweedell

Original Article: Joyner et al. PLoS Pathogens 2019
The Gist of It
Malaria is a devastating disease resulting in more than 228 million cases worldwide and over 400,000 deaths in 2018, mostly in children under the age of 5. Due to the major global public health threat of this disease, there has been intense effort to study it. We know that malaria is caused by the Plasmodium species of parasites. These parasites have a complicated lifecycle, which is one of the reasons it is so challenging to study them. A key feature of some Plasmodium parasites’ lifecycle is that they can form hypnozoites, dormant forms that act like spores, hiding silently in the liver until they reactivate later on to cause what is known as a relapse, or a second infection after the initial “primary” infection is already over. A recent study from scientists at the Malaria Host-Pathogen Interaction Center at Emory University explored how the immune system responds to these relapses. In a rhesus macaque model of infection, they found that, in contrast to primary infections, relapses did not cause the typical symptoms of fever and inflammation. Also, during relapses, the number of parasites in the blood did not get as high as it did during the primary infection. This is at least partly because memory B cells were activated; memory B cells are important immune cells that “remember” a previous infection the body has had, allowing it to fight the pathogen more successfully the second time around. The fact that this immune response led to the absence of clinical symptoms and a lower number of parasites in the blood during relapse seem like a promising result, but upon further exploration of the parasites that were in the blood, the researchers made a worrying finding. While the total number of parasites was lower during relapse, the percentage of these parasites that were gametocytes (the form of the parasite that can be picked up by a mosquito to allow transmission of the pathogen) was actually much higher than in primary infections. This means that although the immune response is doing a good job of limiting the infection, it is not able to stop the production of gametocytes and prevent the further transmission of the parasite. So while your body may be fighting its own infection successfully, this may not translate to a reduction in the number of infections in the community overall. This is an important point to know as we continue to search for strategies to fight this pathogen and prevent its deadly spread.
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During infection with Plasmodium, memory B cells formed as a result of the primary infection can keep parasite numbers low during relapse, but gametocytes are still around and make up a larger percentage of the parasites, meaning the parasite can still be transmitted to other people.

The Nitty Gritty
Joyner et al. established a model of primary infection and relapse using P. cynomolgi in rhesus macaques. The macaques were inoculated with sporozoites to initiate the primary infection. After peak parasitemia in the blood was reached, a single sub-curative dose of artemether was given to reduce but not eliminate the parasites (to prevent the development of severe disease), and then the parasitemia was allowed to rebound. Then the macaques were treated with a curative artemether regimen to clear all blood stage parasites. Subsequently, they were monitored for the occurrence of relapse, which would be due to the emergence of hypnozoites based on this infection and treatment scheme. They observed that relapses were not as clinically severe as primary infections, with anemia, fevers, and thrombocytopenia absent during relapse. Transcriptional analysis of whole blood samples revealed that clusters of genes associated with B cells, T cells, cell signaling, and antigen presentation were upregulated during relapse. By analyzing the peripheral blood mononuclear cells with flow cytometry, they found that during relapse the number of unswitched and switched memory B cells increased. They analyzed the functionality of the antibodies produced by allowing opsonization of Plasmodium-infected red blood cells (iRBCs) with heat-inactivated plasma and testing for phagocytosis by THP-1 cells. They found that the plasma collected during relapse increased the percentage of phagocytosed iRBCs compared with the plasma from naïve animals and compared with the plasma collected during the peak of parasitemia during the primary infection. Finally, the researchers analyzed the effects of the immune response on the parasites during relapse. They found that the overall number of parasites in the blood during relapse never reached the level achieved during primary infection. When they further parsed the data to look at the number of gametocytes specifically, the overall number was also reduced during relapse compared with primary infection. However, the percentage of parasites that were gametocytes was significantly higher during relapse. This suggests that while a strong adaptive immune response is formed that can limit the clinical manifestations of disease, this response will not actually prevent gametocyte formation and subsequent transmission.
Original Research Article: Joyner, C.J., et al. “Humoral immunity prevents clinical malaria during Plasmodium relapses without eliminating gametocytes.” PLoS Pathog 15.9 (2019): e1007974.

How are babies kept safe from mom’s immune system?

Written By: Kaitlyn Sadtler

Original Article: Salvany-Celades et al. Cell Reports 2019
The Gist of It
Why is it that our body rejects transplant organs (even from relatives) but mothers’ bodies are able to safely carry a developing fetus? Nutrients and oxygen travel from the mother to the fetus to support its growth, but normally the mother’s immune cells do not reject this foreign being inside of their own body. Certain kinds of immune cells – known as regulatory T cells (Tregs) – do just as their name suggests, they regulate immune responses. These cells are found at the interface between the fetal and maternal tissue, within the placenta. The placenta is composed of cells from both the mother and the fetus, with blood from both being separated by only a few cells. This allows the oxygen and nutrients from the mother to transfer to the developing fetus, but also provides a site where maternal and fetal cells directly interact. The Tregs here receive signals from fetal cells known as extravillous trophoblasts (“extra” = outside of, “villous” = the fetal blood vessels, and “trophoblasts” = cells from the embryo derived from the same cells that make up the amniotic sac) and also maternal decidual macrophages (“decidua” = lining of pregnant uterus). These cells communicate with the Tregs to prevent immune rejection of the fetus. Salvany-Celades and colleagues described three distinct types of Tregs that are found in this interface. These three cell types can inhibit the activity of both activated helper T cells, which can normally produce proinflammatory cytokines, and cytotoxic T cells, which normally kill cells that are infected by viruses (or donor cells in transplants). As we learn more about the way that these immune cells are working in an optimal setting, we are more aware of signs to look for to identify when something is going wrong; importantly, this also allows us to develop new treatments to help prevent damaging immune responses against developing fetuses.
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Three distinct types of regulatory T cells help train mom’s immune system to prevent it from reacting against the developing fetus.

The Nitty Gritty
Salvany-Celades et al. identified three subtypes of regulatory T cells in the decidual tissues of the maternal-fetal interface in first trimester decidua. These three are divided based on their expression of surface markers: (1) CD25hiFoxP3+, (2) PD1hiIL-10+, and (3) TIGIT+FoxP3dim. These cells were sorted and then tested for their ability to prevent CD4+ and CD8+ effector T cell activation. All three cell types reduced the proliferation of CD4+ effector T cells as determined by CFSE labeling and co-culture and stimulation with anti-CD3 anti-CD28 beads. The first class of Tregs (CD25hi) also inhibited CD8+ T cell proliferation, whereas the other two classes did not. Furthermore, when these classes were isolated from term placenta, they did not have as great of a regulatory capacity as first-term Tregs. The PD1hi Tregs were dependent upon IL-10 signaling, as determined by IL-10R antibody blocking, but this was not required for CD25hi Tregs. When co-cultured with peripheral CD4+ T cells, extravillous trophoblasts (EVTs) and decidual macrophages were able to increase the proportion of FoxP3+ and HELIOS+ Tregs. EVTs and decidual macrophages were also able to induce Tregs in a transwell system, but to a lesser extent than with direct co-culture, suggesting both soluble signals and cell-cell contact are important in the generation of iTregs (induced Tregs) by EVTs and decidual macrophages. Blocking HLA-C on EVTs decreased their ability to promote the differentiation of PD1hi Tregs through cell-cell contact. Overall, this work describes the interaction of three types of Tregs within decidual tissue of first-trimester pregnancies that are educated by both EVTs and decidual macrophages to promote regulation and prevent pathogenic immune responses in pregnancy.
Original Research Article: Salvany-Celades, M., et al. “Three types of functional regulatory T cells control T cell responses at the human maternal-fetal interface.Cell Rep 27.9 (2019): 2537–2547.