Some people can control HIV infection without drugs – can we learn from them to make new therapeutics?

Written By: Kaitlyn Sadtler

Original Article: Gonzalo-Gil et al. eLife 2019
The Gist of It:
HIV-1 infects more than 36 million people across the globe. This virus is tricky to treat because it infects our immune cells (specifically, T cells), it mutates so frequently that it is difficult to make an antibody that will continue to work as the virus changes, and it can hide from immune detection by integrating its genetic material into our genome to form “latent reservoirs” that can re-activate later on. However, in the 2000’s, people who are able to control HIV infection without the use of antiretroviral drugs were first described. Since the discovery of these individuals (called “elite controllers”), researchers have tried to understand what makes them different from the majority of those infected, who cannot control the infection on their own. Understanding this difference would allow us to design therapeutics to target the disease in patients who cannot control it. Researchers from Yale University, the Dallas VA Medical Center, Emory University, the Ragon Institute, and UCSF teamed up to analyze how a set of elite controllers could control HIV-1 infection. They identified that these patients had lower levels of two proteins that mediate inflammatory responses, CCR2 and CCR5, which were not previously described in the context of elite controllers. It has been previously shown that patients who have a mutation in their CCR5 gene called “CCR5 delta” or “Δ32CCR5” cannot contract HIV through sexual transmission. The elite controllers in this study did not have this mutation, but did produce low levels of CCR5 (in addition to CCR2). Family members of elite controllers had the same decreased CCR2 and CCR5 production, suggesting a heritable component to this phenomenon. Further understanding of patients who can control their HIV infection can lead to new therapeutics and drugs to help treat and decrease the HIV epidemic that has spread rapidly since its detection and eventual description in the 1980s.
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Normally, when someone gets infected with HIV, the level of virus in their system or “viral load” peaks early, the immune system fights it off, but then the virus comes roaring back, infecting and killing immune cells. Elite controllers, or “ECs”, are able to keep HIV at bay without anti-retroviral drugs.

The Nitty Gritty:
Gonzalo-Gil et al. evaluated infection and gene expression of Elite Controllers (ECs) and Viremic Controllers (VCs) from the UCSF SCOPE cohort (https://clinicaltrials.ucsf.edu/trial/NCT00187512). They previously identified three patients resistant to R5 viral infection. To evaluate the nature of this resistance, they tested CD4+ T cell infection in vitro and conducted analyses comparing gene expression between patients and direct family members to determine if there was a heritable component for this resistance. They discovered lower expression of the ccr2 and ccr5 genes in both the patients and their family members compared to healthy donors. The chromatin around these genes was open and accessible, but several genes around the ccr2 and ccr5 segment of the chromosome (≈ 500 kB section; 3p21) were also decreased in expression. Chromatin immunoprecipitation sequencing (ChIP-seq) was performed to pull down genes associated with Rpb1 CTD (RNA polymerase carboxy terminal domain).  Levels of both ccr2 and ccr5 were decreased in these patients compared to healthy donors, further supporting a decrease in expression of these genes in the subset of ECs and VCs. With the correlation between gene expression in family members, a heritable nature is suggested, but not understood at this point.
Original Research Article: Gonzalo-Gil, E., et al. “Transcriptional down-regulation of ccr5 in a subset of HIV+ controllers and their family members.” eLife 8 (2019): e44360. doi: 10.7554/eLife.44360.

Aphids have the gall… to sacrifice themselves in a pool of bodily fluids to save their home

Written by: Padmini S. Pillai

Original Article: Kutsukake et al. PNAS 2019
The Gist of It:
If you’ve had a paper cut recently, you may have noticed how quickly your body can repair the damage. Your immune system begins to heal the wound by forming a clot and protecting you from invading bacteria or viruses. The body uses cells called platelets to form a plug to stop the bleeding, and a mesh of fibrin stabilizes the clot to form a scab. Researchers have now demonstrated that insects called aphids do this on a societal level to repair their home and protect their colony from invaders! After an invading insect damages their plant-made nest, called a gall, an army of Nipponaphis monzeni, also called soldier nymphs, head to the breach and release most of their bodily fluids in a process called “body eruption”. Kutsukake and colleagues revealed that this fluid contains two components: cells filled with fat droplets and an enzyme called phenoloxidase (PO), and a liquid that contains the amino acid tyrosine and a protein called repeat-containing protein (RCP). When this fluid cocktail is released at the breach, the cells begin to form a soft plug, and PO turns tyrosine into reactive compounds called quinones. Quinones physically link RCPs together to form a strong mesh that reinforces and hardens the clot. In the process, these altruistic aphids die; some are buried in the forming clot, or get blocked out from the nest, while the rest succumb from the massive discharge of body fluid (even if they are able to crawl back in the gall). Over time, the plant tissue will regrow over the clot and the barrier will be restored. By developing a process akin to our body’s method of scab formation for wound healing, these aphids have evolved to use their immune cells and molecules to sacrifice themselves for the greater good of their colony! As Captain Spock once said, “the needs of the many outweigh the needs of the few.”

 

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Soldier nymphs sacrifice themselves by secreting a cocktail of fluid and coagulants to seal their damaged nest.

The Nitty Gritty:
Soldier nymphs were mechanically stimulated to secrete their body fluid, which was found to contain large globular cells (LGCs) and proteins. Immunoblotting revealed that a predominant protein was the pro-enzyme form of phenoloxidase (PO), which is involved in melanin synthesis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunoblotting, and sequencing revealed a protein with repeats in glycine, serine, histidine, and glutamine, termed repeat-containing protein (RCP). In situ hybridization and immunohistochemistry showed that PO was localized inside LGCs and that RCP was extracellular. PO-related genes were upregulated in LGCs of soldier nymphs. Free tyrosine was abundant in the body fluid. Recombinant PO from Sf9 cells was purified and combined with PO and RCP, which resulted in RCP polymerization and cross-linking and darkening of the solution. Lipid droplets contained within LGCs mainly consisted of triglycerides.
Original Research Article: Kutsukake, M., et al.Exaggeration and cooption of innate immunity for social defense.” Proc Natl Acad Sci USA (2019): 201900917.

Get in line! Using electricity to pattern living cells

Written by: Ritu Raman

Original article: Bajaj et al. Advanced Healthcare Materials 2012
The Gist of It:
Imagine being able to replace a diseased or damaged tissue or organ with new tissue that looked and acted just the same. That is just what scientists are trying to do in the new field of “tissue engineering”! By putting living cells and proteins together bit by bit, like LEGO® building blocks, they hope to build replacement tissues that can safely integrate with your body and help you return to normal, healthy life after an illness or injury. There are many challenges that must be addressed before scientists can do this reliably and for large numbers of patients, though. One of these challenges is being able to control the placement and orientation of very small living cells, a process called patterning, and assemble these cells into precise and complex multi-cellular configurations. A single cell ranges from about 1–100 microns. For comparison, a human hair is usually 17–181 microns thick, so you can imagine how hard it would be to pattern something that small into a reliable 3D shape! Bajaj and colleagues propose one solution to this problem using a phenomenon known as dielectrophoresis (DEP). When cells are placed in a non-uniform electric field generated by electrodes (metal conductors), DEP exerts a force on them that makes them align parallel to the electrode. When you turn off the electric field, the cells lose their alignment. In this study, the authors mixed cells with a light-sensitive liquid solution that could become a solid in response to a laser. They placed the cell-liquid mix on top of electrodes, turned on the electric field to align the cells, and then shined a laser on the solution to turn it into a solid before turning off the electric field. The cells were then fixed in their aligned position inside the solid material. Using this approach, the researchers showed that they could reliably and quickly pattern many cells into complex configurations, creating a new tool that other scientists can use for tissue engineering. I’m so excited to see how people use this in the future! Are you?
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An electric field is used to line up cells into complex patterns. This phenomenon is called dielectrophoresis!

The Nitty Gritty:
Bajaj et al. patterned two types of murine cells, embryonic stem cells and skeletal muscle myoblasts, inside poly (ethylene glycol) diacrylate hydrogels. The pre-polymer solution containing cells was pipetted onto glass slides patterned with electrodes and placed within a stereolithographic 3D printer. Cell patterning was achieved by powering the electrodes with a waveform generator outputting a 10 Vpp, 1–10 MHz sinusoid. Live/dead assays confirmed cell viability in response to DEP-based patterning.
Original Research Article: Bajaj, P., et al. “Patterned three‐dimensional encapsulation of embryonic stem cells using dielectrophoresis and stereolithography.” Advanced Healthcare Materials 2 (2013): 450-458.

What can butterflies tell us about how we might respond to higher temperatures?

Written by: Ken Estrellas

Original Article: Franke et al. BMC Evolutionary Biology 2019
The Gist of It
As winter turns to spring and the temperatures warm in more temperate climates, many humans adapt in some way – changing the clothes we wear, outdoor activities we participate in, and possibly even our sleeping patterns. But what about the other creatures around us – bacteria, mammals,  and insects like the butterfly? Although the effects of worldwide temperature warming on butterflies’ migratory patterns have been well-studied, a recent report from Franke and colleagues looks at a more fundamental change – the effect of temperatures on butterflies’ bodies themselves. In this study, male and female butterflies raised in the same environment were subjected to different temperatures (19°C and 27°C) and different diets (food restriction and unlimited food availability). The researchers observed that varying temperatures had significant effects on the body composition of both male and female butterflies in terms of their mass distribution between the thorax and abdomen (butterflies at cooler temperatures tended to have heavier thoraces but lighter abdomen), as well as their body fat content. Activity of the important protective enzyme lysozyme was reduced with elevated temperature and food restriction, which indicates potentially compromised immune system function. Interestingly, food restriction was not associated with significant effects on overall body mass or fat content but did appear to have effects on thorax/abdomen mass distribution and lysozyme activity. To determine the mechanisms that drive each of these changes, patterns of genetic expression under each of these conditions were analyzed using RNAseq, a technique that sequences the RNA transcripts made from DNA to determine how much of each transcript is present. A total of 659 transcripts were found less often at the elevated temperature, while 430 transcripts were found more often. Many of the genes that were found more often at higher temperatures, such as peroxidase/oxygenase and the cytochrome P450 family, are involved in stress responses and may play a role in tolerance of increased temperatures. These results demonstrate that elevated temperatures can have specific effects on an organism’s gene expression and body composition; while these results are interesting in butterflies, future research in other organisms could potentially provide insights into how all of us might adapt to a constantly warming world.
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At higher temperatures, butterflies showed differences in gene expression (increased oxidative stress genes) and body composition

The Nitty Gritty
In this study, Franke et al utilized the tropical butterfly Bicyclus anyanna to study the effects of elevated temperature, diet, and gender on body composition and genetic expression. Four treatment groups were used: 19°C with food available ad libitum, 19°C with food restrictions, 27°C with unlimited food, and 27°C with food restrictions. The butterflies were monitored over the course of 8 days, then shock frozen at −80°C for further analysis. Statistically significant differences were associated with temperature alone in thorax mass, abdomen mass, thorax/abdomen ratio, fat content, and lysozyme activity. Food restriction alone was associated with significant variations in thorax/abdomen ratio and lysozyme activity. Whole abdomens were used for RNA extraction and subsequent analysis via RNAseq with a de novo assembly process, since a reference genome for B. anyanna is not available. A total of 659 transcripts were shown to be upregulated due to temperature, including those that encode vacuolar H+-ATPase V1 sector, aldehyde dehydrogenase, and trypsin along with genes associated with oxidative defense. In contrast, 430 transcripts were downregulated at elevated temperatures, including those that encode dyneins, aspartyl beta-hydroxylase, a synaptic vesicle transporter SVOP, the chromatin remodeling protein HARP/SMARCAL1, a CUB domain-containing protein, and a chaperonin complex component. Notably, no association was found between components of the heat shock response in this study, likely because the elevated temperature condition of 27°C was still within the range of generally tolerated temperatures for this species. These variances in body composition and gene expression are potentially reflective of differences in energy expenditure, immune function, and oxidative stress responses as a result of elevated temperature. Future studies in organisms could elucidate the effects that overall warming climates might have on influential insect species such as bees, socioeconomically crucial animals such as livestock, and potentially humans.
Original Research Article: Franke, K., al. “Effects of adult temperature on gene expression in a butterfly: identifying pathways associated with thermal acclimation.” BMC Evolutionary Biology 19 (2019): 32.

“Pioneer” bacteria and how they drive the spread of Listeria monocytogenes

Written by: Rebecca Tweedell

Original Article: Ortega et al. eLife 2019
The Gist of It:
Many of us have experienced that unpleasant feeling of food poisoning. One of the pathogens that can cause food poisoning is the bacterium Listeria monocytogenes. This bacterium invades the cells of your intestines and spreads from cell to cell as it multiplies. In people with compromised immune systems, such as those fighting cancer or suffering from autoimmune disease, the infection can eventually expand from the intestines to other organs, causing much more damage than the typical vomiting that comes with food poisoning. L. monocytogenes has a unique way of spreading from cell to cell. It builds a comet tail using a protein called actin that propels it through cells. Once it reaches the edge of a cell, it builds a protrusion that sticks out from that cell and into another one. The neighboring cell then gobbles up the protrusion, pulling the bacterium along with it. A collaboration between researchers at Stanford University and the University of California San Diego recently used video microscopy to watch L. monocytogenes as they spread. Interestingly, they found that not all the bacteria spread the same distance. While many bacteria only spread to cells very close to their original host cell, a few “pioneer” bacteria built much longer protrusions to carry them to much more distant cells. Once they traveled this long distance, the bacteria continued replicating and dividing, creating a new site of infection far removed from the original one. This type of pioneer behavior may improve the bacteria’s chances of establishing a long-term infection, which is bad news for humans. Now that we know about these pioneer bacteria, we can begin to develop ways to stop them and prevent the spread, which could really help those suffering from these infections.
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Individual Listeria monocytogenes use their actin tail to travel different distances and spread from cell to cell in the intestines.

The Nitty Gritty:
Ortega et al. used video microscopy combined with statistical modeling to follow the spreading dynamics of mTagRFP-expressing L. monocytogenes in Madin-Darby canine kidney epithelial cell monolayers. By monitoring the mean squared displacement (MSD) of the bacteria over time, they found a linear increase in MSD (consistent with a random walk model of spread) but also an increasing MSD slope over time (consistent with a model of a fast-spreading organism within a migrating population). They then generated computer models of bacterial spread using equations to model random walk scenarios; these simulations did not match the bacterial spread dynamics observed in culture. The researchers went on to create a computer model that incorporated the existence of “pioneer” bacteria that could travel much farther than the typical bacterium in a single movement. This model assumed diffusive movement of the bacteria, with some having slow diffusivity (standard bacteria) and some having fast diffusivity (pioneer bacteria). When they set the probability of being a pioneer bacterium in the range of 1.4% to 12%, they found that their simulation matched their microscopy data. Importantly, they extended these simulations to model in vivo spread kinetics. This models predicts that the presence of the pioneering phenotype contributes to an increased chance of persistence during infection of the intestinal epithelium and of achieving a “steady state stable” condition in which the bacterial growth is kept in check by the host immune system so as not to cause critical illness, but the bacteria continue to replicate and shed into the feces for dissemination.
Original Research Article: Ortega, F.E., et al. “Listeria monocytogenes cell-to-cell spread in epithelia is heterogeneous and dominated by rare pioneer bacteria.” eLife 8 (2019): e40032. doi: 10.7554/eLife.40032.