Going to places where research has its impact: HIV in KwaZulu-Natal

*SPECIAL ISSUE*

Written By: Kaitlyn Sadtler

Often, scientific researchers are stereotyped to spend their life in the lab and have little experience of the world outside of their academic bubble. To an extent, many of us younger scientists can relate to this. Our goal is to put our head down and get our research done, no amount of time in lab is enough for us to get done what we want to do in lab. So many experiments, so little time. However, when we are doing research to develop therapeutics, then we really need to understand the needs of the end user. What is the point of a therapeutic that a patient would never use?
Some moments in life we get the opportunity that changes our view and really orients us in the larger scheme of things. Gives us that “big picture” feeling. Recently, myself, and another one of our ICEBERG writers, Padmini Pillai, were invited to attend an HIV Conference in South Africa by Bruce Walker (director of the Ragon Institute of MGH, MIT & Harvard). Along with this conference we had the amazing experience of meeting participants from two transformative programs: FRESH and ITEACH.
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Women of FRESH. All enrolled as HIV-negative, they are taught about HIV, how to avoid contracting the virus, and given access to computers and career development.

FRESH: Females Rising through Education, Support, and Health. This program focuses on young, low-income women in the former township of Umlazi in KwaZulu-Natal, South Africa. Here, the incidence of HIV infection for this population of women can reach 60%. That is 3 people infected in every 5. These women have lived with HIV being a constant threat for their entire lives. When we visited the FRESH program, situated in a shopping mall in Umlazi to be more approachable and less stigmatized than clinics or hospitals, we met a group of young women that were so well spoken and enthusiastic to learn from people doing research on HIV, that you could see them on the street in Boston, or San Francisco, and assume they were a local. The FRESH program gave these women education on how HIV is transmitted and what the different drugs did – PrEP for preventing infection versus ARVs for treating HIV infection. Furthermore, the program taught them computer skills and promoted their transition into schooling or employment. With this program, the women (all HIV negative when they join the program) provide weekly blood samples that allow for the quick detection of HIV before acute viral infection symptoms arise, and rapid treatment, as well as increasing our understanding of what occurs in the very early stages of HIV infection. When we were chatting with them, they told us things they liked and didn’t like about HIV prevention and treatment. For example, the sound of the pill bottle is stigmatized, so if they carry PrEP, or someone sees them take a PrEP pill, as opposed to knowing that she is trying to prevent HIV, they assume she is taking ARVs and thus is HIV positive. Also, the pill is too big, they were wondering if they could have PrEP in a powdered drink as opposed to a pill. Things that work for therapeutics in the USA or Europe, might have unforeseen barriers in countries where they are needed the most. In these areas of South Africa, treatments and prevention needs to be discrete, simple, and require minimal visits to the doctor’s office or clinic.
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Traditional Healers, or Sangomas, welcoming us to their town.  Some of these women were healers when the HIV epidemic began.

ITEACH: Integration of TB in Education and Care for HIV. In many cultures, there are elements of traditional healing – from prayer, to herbal remedies, to that thing Mom told you helped colds and you swear by it. In South Africa, traditional Zulu healers call on their ancestors to determine how to help treat illness. This form of healing can be seen as a bit taboo in hospitals, where patients can receive judgement for seeking traditional healing and can be told to not see healers anymore by their doctors at clinics. This leads to patients either not telling their doctors if they are seeing healers (and thus doctors not knowing what herbal remedies might be taken) or the thought that they must pick either clinical medicine or traditional healing. The ITEACH program integrates modern treatments for HIV and TB with traditional healing. Sangomas (healers) are taught to be able to identify the signs of HIV, and even perform blood tests in their huts, and then refer patients that test positive to clinics to receive antiretroviral treatment. These healers are critical members of the community, and are a tremendous force in the fight against HIV and TB.
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Research labs in KwaZulu-Natal: These scientists are working on HIV in the heart of the epidemic.

Perspective and real-world knowledge are integral players in the development of viable and impactful therapeutics. Researchers, especially young scientists who have spent the majority of their adult life thus far in the lab and in lectures learning scientific principles and practice, must also learn the principles and practice of other cultures – such as stigmas around disease and community organization – to properly design therapeutics that will treat those that most need them. Furthermore, such perspective can change our viewpoint on day-to-day life. While we were in South Africa, we saw a screening of “Yesterday” a movie about HIV in rural South Africa by Anant Singh. One quote struck me, by the lead character who was a young mother suffering from HIV infection, “I am not brave, this is just the way things are.” When I met the women from the FRESH program, I was inspired by them, but for them this was just how life was. Scientific research has the potential to change infectious diseases, what once was an immediate death sentence with no treatment in the 1980s, is now manageable with antiretrovirals. And hopefully, one day, women like those in the FRESH program, can have a life where “the way things are” isn’t a day to day question of whether or not you will be infected by HIV.
Learn More:
FRESH http://www.ragoninstitute.org/fresh-program-combines-basic-science-with-social-benefits-for-women-at-risk-of-hiv/
iTEACH http://www.ragoninstitute.org/international/iteach/

Your immune system can determine how long you wear braces!

Written by: Ken Estrellas

Original Article: Wang et al. Scientific Reports. 2018
The Gist of It: 
For many people, wearing braces can be an annoying and sometimes painful experience. Depending on how quickly teeth shift and settle in response to the push and pull of the wires or molding, treatment can last 2-3 years or longer. Researchers at Nanjing Medical University’s Department of Orthodontics have identified something other than teeth and gums that can affect how well braces work: the immune system.
In this study, rats and mice had coil springs attached to their teeth to simulate treatment with braces. In addition to braces, some mice and rats also had a corticotomy, a surgical procedure that involves cutting into a tooth to help it move into place when treated with braces. As expected, teeth treated with both corticotomy and braces shifted more easily than teeth treated with braces alone. During the 42 days while the braces were in place, more osteoclasts, cells that absorb bone tissue and help with repair and remodeling, were found in teeth treated with corticotomy and braces than in teeth treated with braces alone. Additionally, more macrophages, a key component of the immune system, were present in corticotomy-treated teeth with braces, and different types of macrophages that act in different ways and have different functions were present at various points in time. These macrophages seem to play a key role in orthodontic remodeling: mice that received these orthodontic treatments (braces ± corticotomy) had decreased levels of tooth movement when all of their macrophages were removed. Overall, this study demonstrates that the immune system can affect how well braces work. These findings could help orthodontists determine what might cause braces to be ineffective in some patients and could guide treatment decisions to ensure patients’ teeth realign properly.
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The immune system affects everything – even how long you might end up wearing braces!

The Nitty Gritty:
To determine the immunological factors associated with orthodontic tooth movement (OTM) and the potential additive effects of corticotomy (CO), Wang et al. used nickel titanium coil springs to ligate the left maxillary first molars to maxillary incisors in adult Wistar rats (at a force of 60 g) and C57BL/6 mice (at a force of 30 g), simulating orthodontic treatment (TM). One set of rats and mice was subjected to vertical CO via supraperiosteal incisions in the left maxillary first molar. Statistically significant increases in tooth movement were observed in rats treated with CO + TM compared to rats treated with TM at several time points over the course of 42 days. Significantly increased osteoclast activity and macrophage infiltration were also observed at several timepoints, as well as increased expression of CD11b and CD68 in rats’ left maxillary first molar palatal gingival tissue. Analysis of bone marrow-derived macrophages from the gingival tissue of mice treated with CO ± TM by ex vivo RT-PCR to determine cytokine expression and western blotting to quantify the levels of p65 and phospho-STAT3 protein revealed a correlation between CO treatment and activation of the NF-κB and JAK–STAT pathways. Immunohistochemistry of rat gingival tissue revealed significantly greater proportions of CD86+ cells in the earlier stages of treatment and greater proportions of CD163+ cells in later stages of treatment, highlighting a change in macrophage polarization. Flow cytometry and RT-PCR analysis of rats’ gingival tissue also revealed dynamic changes in cell surface marker and gene expression over time. Most notably, mice subjected to macrophage depletion via clodronate liposome administration throughout TM ± CO treatment demonstrated significantly decreased levels of orthodontic tooth movement; these mice also displayed macrophage depletion by flow cytometry analysis of spleen and gingival tissue across all treatment groups and timepoints. Overall, this study helps elucidate the heretofore undefined “regional acceleratory phenomenon” (RAP) associated with CO and could potentially lead to the development of diagnostic assays to identify the causes of poor orthodontic performance and identify cases with a need for CO in addition to braces.
Original Research Article: Wang, Yan, et al. “Macrophages mediate corticotomy-accelerated orthodontic tooth movement.” Scientific Reports 8, Article Number: 16788 (2018).

“Good” bacteria can chop up gluten and help those with celiac disease

Written By: Rebecca Tweedell

Original Article: Sakurai et al. Beneficial Microbes. 2018.
The Gist of It:
With all the diet crazes out there today, many restaurants are now advertising a variety of gluten-free options. Some people simply choose to avoid or cut back on gluten as a weight-loss strategy, but some people have to avoid any and all gluten because they are basically allergic to it. This “allergy”, known as celiac disease, leads to inflammatory responses in the gut if the person eats gluten. There are also several other lesser-known conditions that can cause similar reactions to other foods. In people with these conditions, specific food-derived peptides, or chopped up pieces of the proteins within the food, can cause the inflammation, resulting in not only the outward symptoms of chronic diarrhea, upset stomach, bloating, and cramping, but also in internal long-term damage to the digestive system. One major way to prevent the damage would be to destroy these peptides before they even have a chance to cause the inflammation. This is an area where bacteria can actually be good and help out. Bacteria have peptidases, or a type of machinery that can cut up peptides, that we as humans don’t have. Part of the job of the gut microbiome, or the population of microbes that are always living inside your digestive system, is actually to help us break down the foods we eat using these peptidases. Recent work by Sakurai and colleagues at the Morinaga Milk Industry Co., Ltd. In Japan has focused on the peptidases from Bifidobacterium, which is often found under normal conditions in the human microbiome. Bifidobacterium are particularly common in infants, and the specific types of Bifidobacterium living in our microbiome changes as we age. The researchers found that the types of Bifidobacterium isolated from infants were better at breaking down certain food-derived peptides than the types of Bifidobacterium isolated from adults and elderly people. This highlights the possibility that we could use the types of Bifidobacterium normally found in infants as probiotics, like the good bacteria you often see advertised in yogurts these days, to help people with celiac disease and other similar disorders avoid such negative reactions to foods. This could be an important step forward in the fight against these kinds of food intolerance.
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Bifidobacterium can chew up the gluten peptide, keeping people with celiac disease from getting stomach aches and digestive system damage.

The Nitty Gritty:
The researchers screened strains of Bifidobacterium isolated from infant (aged 0-1 years), adult (aged 20-60 years), and elder (aged > 100 years) human specimens, as well as various animal specimens, for peptidase activity. Using a fluorescent dipeptidyl peptidase (DPP) assay with the substrate for dipeptidyl aminopeptidase IV (H-Gly-Pro-AMC), they found that all 18 strains tested had some level of activity, with higher activity in the infant-derived bifidobacteria, including Bifidobacterium longum subsp. longum, B. longum subsp. infantis, B. breve, and B. bifidum. The researchers further characterized the ability of the strains to hydrolyze various food-derived peptides (casomorphin-7 from human milk [hCM-7], casomorphin-7 from bovine milk [bCM-7], and α-gliadorphin-7 from wheat gluten [GC-7]) using multiple reaction monitoring techniques combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). LC-MS/MS allowed them to quantify how much of the intact peptide remained after incubation with the bacterial strain. They found that some of the B. longum subsp. infantis and B. bifidum strains could degrade hCM-7, bCM-7, and/or GD-7. Based on these findings, they performed a subsequent screen on 29 strains of B. bifidum isolated from infant, adult, and elder human specimens. All 29 strains had DPP activity, and 20 of the 29 had 100% degradative capability against hCM-7. While degradative capabilities were generally lower against bCM-7 and GC-7 than against hCM-7, one stain, MCC1319 (from an infant specimen), showed ≥ 50% hydrolysis of bCM-7, and two strains, MCC1135 (from an infant specimen) and MCC1870 (from an elder specimen), showed ≥ 50% hydrolysis of GC-7.
Original Research Article: Sakurai, T, et al. “Degradation of food-derived opioid peptides by bifidobacterial.” Beneficial Microbes 9.4 (2018): 675-682.

Our immune system splices together “Frankenstein Proteins”

Written by: Kaitlyn Sadtler

Original Article: Faridi et al. Science Immunology 2018.
The Gist of It:
When we are checking out at the grocery store and the cashier rings up the produce, the scanner doesn’t read the whole name of the item we are purchasing – it just reads the short barcode that tells the register what item is being scanned. You can think of our immune system in the same way. Instead of seeing the whole bacteria and saying “yup, that’s Staphyloccocus aureus” it looks at the “barcodes”. These peptides, or parts of proteins, that act as the barcode for the pathogen are created when our immune cells (specifically cells called macrophages and dendritic cells) degrade the viral or bacterial proteins into small pieces that can be presented to your body’s barcode-readers, known as T cells.
Recently, researchers from Monash University in Australia discovered that our body actually can create a large number of “Frankenstein” cis or trans-spliced peptides. This means that pieces from two different parts of one protein or even pieces from two different proteins were combined to form one barcode. These types of peptides were thought to be quite rare, but they have historically been difficult to detect due to their remixed nature. Each full protein is made by reading a specific sequence in your genome, much the same way we can read the alphabet from A to Z. We mix up these letters to form words in much the same way these protein pieces are combined to form these Frankenstein peptides. For example, if you were to read the alphabet from start to finish, you would never find the word “the”; similarly, if you were to read your genome from start to finish, you would never find these combined protein pieces. The team created an analysis that was able to predict whether or not these Frankenstein peptides were formed from (1) normal proteins, which are called “linear” peptides, (2) protein pieces cut from two parts of the same protein, which are called “cis” peptides, or (3) protein pieces cut from two different proteins, which are called “trans” peptides. Identifying these Frankenstein peptides will increase our understanding of how our body recognizes infection, as well as how some people develop autoimmune conditions. Furthermore, if we can identify these peptides, we will increase our arsenal to fight infection, create vaccines, and treat autoimmune disorders.
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Our immune system recognizes proteins from viruses and bacteria like grocery scanners recognize barcodes. More frequently than we thought, these barcodes can be spliced together!

The Nitty Gritty:
Mass spectrometry (MS) data from 17 human cell lines were used to identify different HLA-bound peptides (p-HLA). Using PEAKS Studio 8.5 software, Faridi et al. first identified linear p-HLA by matching against the human reference proteome. Those p-HLA that did not map to the human proteome were considered to be either linear peptides that fell outside of their FDR criteria, cis or trans spliced peptides, or peptides with (currently) no potential explanation. These p-HLA were run through their novel hybrid-finding algorithm with assumptions that cis-spliced p-HLA would be more likely than trans, both of which would be less likely than a linear p-HLA. The outcomes of this software were then merged with the original proteome dataset and the analysis was conducted on the MS data again to identify fractions of p-HLA peptides that were cis or trans spliced. Through their analyses, they discovered that 12-44% of p-HLA were either cis- or trans-spliced peptides. Furthermore, the proportion of total non-linear p-HLA that were trans-spliced was quite large – up to 25% of total p-HLA in some monoallelic cell lines. Several of these p-HLA were validated through crystal structures of cis- and trans-spliced p-HLA in the binding groove of HLA. Faridi et al. went on to show that there was a preference for spliced peptides in the 9-10-mer range, with combinations of splice fragments being most likely 4 + 5 amino acids.
Original Research Article: Faridi, Pouya, et al. “A subset of HLA-I peptides are not genomically templated: Evidence for cis-and trans-spliced peptide ligands.” Science immunology 3.28 (2018): eaar3947.