Thursday, August 17, 2017

Gonococcus superbug: Current Status

Photo 1: Neisseria gonorrhoeae.
Neisseria gonorrhoeae or what is commonly known as the gonococcus is sounding serious alarms all over the globe. In a post almost an year ago, I talked about how gonococcus is slowly rising to the status of true superbug. Gonococcus is responsible for a sexually transmitted disease called as Gonorrhea. Decades ago, this was absolutely treatable with a simple penicillin. However, they have now acquired several genes that makes it more untreatable.

Table 1: Resistance pattern of Gonococcus. Source
Here is the summary of current issue based on WHO reports in June. WHO has estimated that nearly 78 million people are infected with gonococcus. There are countries that are reporting their annual statistics and some countries that dont. The current recommended regimen for gonorrhea treatment is a combination of azithromycin and ceftriaxone. Table 1 shows a summary of number of countries in different WHO regions reporting gonococcal isolates with resistance to azithromycin and ciprofloxacin, and decreased susceptibility or resistance to extended-spectrum cephalosporin (Cefixime and/or ceftriaxone) for at least 1 year from 2009 to 2014. It should be noted that the report is based on data from 77 countries. Most African nations where the percentage of gonorrhea is expected to be higher has not reported. Interestingly, WHO has reported 3 confirmed cases of gonococcus that are resistant to all the anitbiotics tested. 

“These are cases that can infect others. It can be transmitted. And these cases may just be the tip of the iceberg, since systems to diagnose and report untreatable infections are lacking in lower-income countries where gonorrhea is a ctually more common.”
-Teodora Wi (Department of Reproductive Health and Research, WHO)

“To control gonorrhoea, we need new tools and systems for better prevention, treatment, earlier diagnosis, and more complete tracking and reporting of new infections, antibiotic use, resistance and treatment failures. Specifically, we need new antibiotics, as well as rapid, accurate, point-of-care diagnostic tests – ideally, ones that can predict which antibiotics will work on that particular infection – and longer term, a vaccine to prevent gonorrhoea.”
-Marc Sprenger, (Director of antimicrobial resistance, WHO)

By now you understand that gonorrhea is a serious issue and needs to be addressed qucikly. Vaccine is an excellent choice. But vaccine research in gonorrhea isn't so great and there are no candidates in sight that are ready to be launched. Howewer, there is a silver lining.

A study was recently published which retrospectivly looked into vaccine effectiveness of outer membrane vesicle meningococcal B vaccine (MeNZB) in a case-control study of patients at sexual health clinics aged 15–30 years. They found that the gonorrhea rate among teens and young adults who had received a meningitis B vaccine during an emergency campaign in the early 2000s was significantly lower than the rate seen in people of the same age who weren’t vaccinated. The estimated vaccine effectiveness of MeNZB against gonorrhoea was about 31%. Of course its a chance observation and some form of clinical trial needs to be done to have more credibitily for the claim and get a more realistic estimate. There are also similar reports of decline in gonorrhea rates follwing meningococcal vaccine in Cuba, and Norway.

There is a lot of effort currently in trying to come up with new antibiotics especially ones that can target such superbugs. A compound referred to as closthioamide has been shown to have promising results against drug resistant gonococcus.

Fig 1: Structure of Clostioamide.
Source
Closthioamide (CTA) was first discovered and isolated from Anaerobic Bacterium Clostridium cellulolyticum in 2010 which was initially tested as a possible compound against multi drug resistant Staphylococcus. It functions through attacking DNA gyrase. In brief, the researchers tested 149 strains isolated from patients, 8 WHO reference strains of of N. gonorrhoeae and 4 commensal Neisseria strains were tested. CTA performed really well against 146/149 (98%) of clinical gonococcal strains at ≤0.125mg/L. The study also noted that two N. perflava strains had the highest CTA MIC (>1 mg/L).

As the senior author of the study John Heap comments, "The imminent threat of untreatable antibiotic-resistant infectious diseases, including gonorrhoea, is a global problem, for which we urgently need new antibiotics. This new finding might help us take the lead in the arms race against antimicrobial resistance. We believe there are many undiscovered antibiotics out there in nature, but they are difficult to find and test. For example, the bacteria which produce closthioamide naturally make only tiny amounts that are not enough to test or use, so we had to chemically manufacture it ourselves by mimicking its natural structure. The next step will be to continue lab research to further assess the drug's safety and effectiveness. Despite showing tremendous promise, it will be a number of years before, and if, we can use the drug in real life human cases."

As of now there is no clear answer as to how to tackle the globally spreading true superbug "gonococci" is to be controlled. Perhaps the best method is the same as standard STD prevention methods.

References:

1. Wi T, Lahra M, Ndowa F, Bala M, Dillon J, Ramon-Pardo P et al. Antimicrobial resistance in Neisseria gonorrhoeae: Global surveillance and a call for international collaborative action. PLOS Medicine. 2017;14(7):e1002344. 

2. Petousis-Harris H, Paynter J, Morgan J, Saxton P, McArdle B, Goodyear-Smith F et al. Effectiveness of a group B outer membrane vesicle meningococcal vaccine against gonorrhoea in New Zealand: a retrospective case-control study. The Lancet. 2017; doi: 10.1016/S0140-6736(17)31449-6. [Epub ahead of print]

3. Miari V, Solanki P, Hleba Y, Stabler R, Heap J. In vitro susceptibility to closthioamide among clinical and reference strains of Neisseria gonorrhoeae. Antimicrobial Agents and Chemotherapy. 2017;:AAC.00929-17.

4. Lincke T, Behnken S, Ishida K, Roth M, Hertweck C. Closthioamide: An Unprecedented Polythioamide Antibiotic from the Strictly Anaerobic Bacterium Clostridium cellulolyticum. Angewandte Chemie. 2010;122(11):2055-2057.

Wednesday, August 09, 2017

Scientists find a quick method to get Monoclonal Antibodies of interest.

A lot of new emerging infectious diseases are no on global radar and that highlights how unprepared we are in fighting it. Most of these are geographically limited and terminate with a few countable number of cases. The one's like Influenza variants, Zika and Ebola have been more rampant. Though a short term solution is to administer antibiotics or quarantine, the best approach is to vaccinate. The centre of this whole problem lies in B cells.

Fig 1: B cell activation. Source: Kuby Textbook; 5e
I need to visit back the B cell activation pathway. B cells are a type of lymphocytes that is involved in making antibodies. B cells by nature are inactive and have to be activated specifically. The individual lineage of B cells can make antibodies against a specific antigen. These inactive B cells which are competent enough to start making antibodies, provided they have the right signal, is called as immunocompetent B cells.

Depending on the nature of the antigen, there are two modes of B-cell activation. TH cells dependent (TD) and TH cell independent (TI). There are 2 types of signals that are required as membrane events, to activate a B cell. The first activation signal is an antigen binding to B cell receptors (BCRs). Once bound, the antigen is internalized by receptor-mediated endocytosis, digested, and complexed with MHC II molecules on the B cell surface. The second activation signal (also referred as the costimulatory signal) is CD40/CD40L interaction. See Fig 1. Once activated they go on to mature and convert to B cells which start making antibodies. There is some evidence that this is not completely true and there are more signals in the interaction. For example, TLR is possibly the third signal. 

There have been several previous attempts in the laboratory to replicate this process in the laboratory condition. Previous studies have shown that patient-derived B cells when treated with CpG oligonucleotides, they stimulate every B cell in the population. CpG oligonucleotides are short single-stranded synthetic DNA molecules that contain a cytosine triphosphate deoxynucleotide followed by a guanine triphosphate deoxynucleotide. The CpG is mainly recognised by TLR 9, which is expressed in B cells and Plasmacytoid dendritic cells and is thus an excellent immunostimulant. Antigen-dependent activation of B cells in-vitro is difficult to achieve result because the wide haplotype variation of MHC IIs necessitates the use of unique T cells specific to a particular MHC II to activate B cells in vitro.  This problem was solved by the team led by Facundo Batista, from the Francis Crick Institute in London based on which the current paper is built.

The researchers started with coated streptavidin polystyrene nanoparticles containing a mixture of biotinylated anti-κ antibody and the TLR ligand CpG. The team showed that by treating the patient derived B cells with the coated nanoparticles and the appropriate antigen. In short, CpG oligonucleotides are only internalized into those B cells that recognize the specific antigen coated, and these cells are therefore the only ones in which TLR9 is activated to induce their proliferation and development into antibody-secreting plasma cells. 

The team has shown that the results are replicable when done with different bacterial and viral antigens (such as tetanus toxoid and proteins from several strains of influenza A, HIV gp120). The studied showed specifically that in vitro stimulation of memory B cells with particulate antigen-CpG selectively enriched the frequency of CD27hi/CD38hi antigen-specific plasma cells irrespective of the nature of the antigen that was chosen. So technically in proof, this method can be applied to any antigen. Further, it was achieved in a very short time, almost a week.

This novel method is much superior to several other methods such as phage display, EBV immortalization, yeast display, and humanized animal models since it doesn't rely on a large scale screening and identification. Basically, this method allows for selective stimulation of memory B cells from healthy donors, leading to proliferation and differentiation into plasma cells that produce antigen-specific antibodies, even in antigen-naive donors (They demonstrated by showing you could develop antibodies against HIV from cells derived from HIV negative donors), which means making therapeutic vaccines (Antibodies) could be very fast.

As Facundo Batista explains, "Specifically, it should allow the production of these antibodies within a shorter time frame in vitro and without the need for vaccination or blood/serum donation from recently infected or vaccinated individuals. In addition, our method offers the potential to accelerate the development of new vaccines by allowing the efficient evaluation of candidate target antigens."

References:

Irene Sanjuan Nandin, Carol Fong, Cecilia Deantonio, Juan A. Torreno-Pina,Simone Pecetta, Paula Maldonado, Francesca Gasparrini, Jose Ordovas-Montanes, Samuel W. Kazer, Svend Kjaer, Daryl W. Borley, Usha Nair, Julia A. Coleman, Daniel Lingwood, Alex K. Shalek Eric Meffre, Pascal Poignard, Dennis R. Burton, and Facundo D. Batista. Novel in vitro booster vaccination to rapidly generate antigen-specific human monoclonal antibodies. The Journal of Experimental Medicine, 2017 DOI: 10.1084/jem.20170633.

Eckl-Dorna J, Batista F. BCR-mediated uptake of an antigen linked to TLR9 ligand stimulates B-cell proliferation and antigen-specific plasma cell formation. Blood. 2009;113(17):3969-3977.

Tuesday, July 25, 2017

Dopamine says "Make antibodies"

Photo 1: Lymphatic system
in brain. Source
For a long number of years nervous system and immunological system have been seen as two separate systems. In the last couple of decades this idea has been strongly questioned. Central nervous system which has been assumed to be devoid of immune activity is now known to harbour immune system of its own (Link). Their location was quite close to prominent blood vessels. There has been some proof that neural system could in part regulate neural activity also (Link). In 2015, Louveau et al reported that the brain has a lymphatic system of its own. The anatomical discovery was surprising since the vessels’ were hidden in a location deep within the brain. Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), is a marker of lymphatic system. LYVE1 immunostaining of whole-mount meninges is shown in Photo 1 showing distribution of brain lymphatics.

There are several papers that have established that neuroimmune interactions are 2 way. Classical nervous system molecules such as dopmaine can act on immune cells. For example, T cells express several dopamine receptors (DARs). It has been established that stimulation of specific DARs on Dendritic cells and T cells, influence CD4+ T cell differentiation into Th1 or Th17 inflammatory cells. Dopamine receptors are universally expressed in T cells, dendritic cells (DCs), B cells, NK cells, neutrophils, eosinophils, and monocytes. Fig 1, shows an example of established pathways on how neurotransmitters affect T cell response. Ofcourse, the reverse is also true, though the phenomenon is less well studied. A really good example is cytokines. IL-6 is now highly implicated as a Neuropoetin (Stimulate Neuronal growth), though mechanism is not clearly defined.

Fig 1: Neurotransmitter-mediated regulation of T cell response. Source
So far, so interesting. There is convincing evidence that dopamine receptor is important. But having a receptor is one thing. There has been some research earlier suggesting that the immune cells can themselves also make dopamine and it has not been clear as to what is the role. And thats the new story.

Making an antibody is a very tightly regulated process. When the immune system encounters a foriegn molecules the T cells delivers signal to B cells in a complicated molecular process. In germinal centres, highly mobile T cells and B cells specific for the same pathogen can directly interact with each other through the formation of dynamic specialized surface structures called T–B immunological synapses. In a new study by Papa etal, showed that the Follicular helper T cells or TFH (They are also known as Follicular B helper T cells) use dopamine as cargo loader for immune molecules which mediates a T-B synapse which triggers B cell maturation.

Fig 2: Graphic model of the proposed positive feedback
between human TFH and germinal centre B cells.
Source
The first experiment goes on to show that Chromogranin B (CGB) is present in human tonsils, spleens, and lymph nodes and determined that the T cells isolated from these samples icontained granules filled with dopamine. Using a method called live-cell RNA detection CGB was shown to be present in high quantities of human germinal centre TFH cells but not so much in other T cells. Subsequent experiments showed that dopamine was not really there in detectable amounts in cells other than TFH cells. Next set of experiments showed that human TFH cells released dopamine on stimulation by germinal-centre B cells and it upregulated the ICOSL on the cell surface of germinal-centre B cells. This process was also shown to enhance accumulation of CD40L and chromogranin B granules at the human TFH cell synapse and increases the synapse area. The tests also showed that the process could be blocked by haloperidol and a DRD1 specific antagonist SKF83566, thus narrowing down the receptor to DRD1. Based on the experimental findings, the authors proposed an interaction model, shown in Figure 2. According to an explanatory accompanying paper, it has been hypothesised that TFH cells have a very stringent requirement for efficiency and specificity at the immunological synapse, which explains the finding that dopamine is used by human TFH cells, but not by human T cells of other subclasses.

So now I have some questions. Is there a possible mechanism where dopamine from a neuron stimulates B cells? Does this finding explain why in certain dopamine related disorders such as schizophrenia (where there is presumably an increased dopamine actvity) have an increased autoimmune phenomenon. As Hai Qi speculates, "When disease characteristics or treatment options are associated with changes in dopamine, the possible involvement of, and implications for, antibody- mediated immunity should be considered".

As Papa the lead author comments, “These particles were previously thought to only exist in neurons in the brain and we think they are, potentially, an excellent target for therapies to speed up or dampen the body’s immune response, depending on the disease you’re dealing with. Like neurons, specialised T cells transfer dopamine to B cells that provides additional ‘motivation’ for B cells to produce the best antibodies they can to help to clear up an infection. The human body has developed an advanced form of protection against bacteria, viruses and other foreign bodies that relies on the immune system".

References:

Papa I, Saliba D, Ponzoni M, Bustamante S, Canete P, Gonzalez-Figueroa P et al. TFH-derived dopamine accelerates productive synapses in germinal centres. Nature. 2017;547(7663):318-323.

Qi H. Immunology: Nervous crosstalk to make antibodies. Nature. 2017;547(7663):288-290.

Friday, July 21, 2017

nCD64 as a marker of Sepsis

Several times in my blogs, I have talked about how important it is to make a diagnosis at the fastest turn around time possible. In an attempt to miniaturise the testing platform and obtaining faster results, several technologies have been tested. In context with infections, genome detection and sequencing based technologies are increasingly becoming better and more accessible. Another example is pathogen specific molecular marker detection method on which a good lot of R&D is invested. MALDI-TOF is an excellent example.

Fig 1: Hospitalisation rates for sepsis or septicemia.
Sepsis is a serious issue. Any clinical microbiologist who works in association with the hospital knows the seriousness of sepsis. The terms "Sepsis" and "Septicemia" both refer to a bloodstream infection. Though in a strictly technical sense they mean two different things, they have been interchangeably used in literature and was widely accepted as similar. The earlier definition of sepsis was based on the idea that it is a systemic response and was thus assessed using a systemic inflammatory response syndrome (SIRS) criteria. To date, there is no clear definition of what sepsis is though it is generally agreed that it means circulating pathogen in blood. The diagnosis is based on evidence of fever, respiratory rate and abnormal total WBC count followed by bacterial identification from blood culture. There are no global estimates of sepsis prevalence. Available estimates suggest a range of <1% in a population. However,  there is a significant trend observed everywhere as shown in Fig 1.

In most parts of the globe, a prediction of sepsis is made based on markers such as C reactive protein and procalcitonin levels. Many studies have attempted to come up with a marker. Some of the well-researched markers of sepsis include triggering receptor expressed on myeloid cells-1 (TREM-1), azurocidin, CD64, CD11b etc.

Fig 2: Process schematic of the differential expression-based
cell-counting technology. Source
Studying these markers in the laboratory is not the big deal, since instruments such as Flow cytometers and other sophisticated equipments can do it. But they are not ideal for POCT (Point of care testing). In 2015, this problem was addressed by developing a POCT equipment based on microfluidics. The same group has now come up with improvements in design. The microfluidic biochip is capable of enumerating leukocytes and quantify neutrophil CD64 (nCD64) levels from 10 ml of whole blood without any manual processing. The tech uses whole blood (10ml) which is pumped into the biochip along with lysing and quenching buffers, to lyse erythrocytes. Cells are electrically counted and differentiated based on size using microfabricated electrodes. The CD64+ cells get captured based on their CD64 expression level. The difference in the cell counts is used to calculate nCD64 expression level. See Fig 2.

The authors claim that this technology can have profound results since the assay takes about 30 min and has scope for further improvement. That would be something really usefull to clinicians as a bedside tool for identifying sepsis.

References:

Mervyn Singer et al. The Third International ConsensusDefinitions for Sepsis and Septic Shock (Sepsis-3). JAMA. 2016;315(8):801-810. doi:10.1001/jama.2016.0287

Mayr F, Yende S, Angus D. Epidemiology of severe sepsis. Virulence. 2013;5(1):4-11.

Wang X, Li ZY, Zeng L, Zhang AQ, Pan W, Gu W, Jiang JX. Neutrophil CD64 expression as a diagnostic marker for sepsis in adult patients: a meta-analysis. Crit Care. 2015 Jun 10;19:245. doi: 10.1186/s13054-015-0972-z.

Hassan U, Reddy B Jr, Damhorst G, Sonoiki O, Ghonge T, Yang C, Bashir R. A microfluidic biochip for complete blood cell counts at the point-of-care. Technology (Singap World Sci). 2015 Dec;3 (4):201-213. DOI: 10.1142/S2339547815500090

Hassan U et al. A point-of-care microfluidic biochip for quantification of CD64 expression from whole blood for sepsis stratification. Nat Commun. 2017 Jul 3;8:15949. doi: 10.1038/ncomms15949.

Monday, July 03, 2017

Lab series #17: Labelling methods for Quantitative Proteomics by MS

In an earlier post, I have talked about the principle of how a mass spectrometry works (Link) and how proteomics by sequencing is done using MS (Link). I had a few readers who suggested the idea that I have talked about MS-based shotgun sequencing but proteomics could be done even without sequencing. For example, MALDI-TOF analysis can tell about the protein identity which doesn't involve sequencing. This is absolutely true. However, such assays are now nearly outdated and sequence information can give us a lot more insight than just predicting protein based on the m/z values. In the earlier post, I ended with a note saying that I will revert back to the topic and talk about proteogenomics, targeted proteomics and quantitative proteomics. In this post, I will talk about labelling methods for quantitative proteomics or sometimes referred to as differential proteomics. If you have not read my earlier posts on MS, I strongly recommend that you read them first.

Let us build an example scenario. You want to learn what are the changes that occur in the cell after a virus infection. The most likely scenario in terms of proteome would be certain proteins will have increased expression and certain will have decreased expression, as a result of interaction with a virus. If you could find out what those proteins are, then there is a good chance that you could predict the pathways that have been disrupted. But for identifying what is the fold change, we have to quantify each protein. In a traditional assay like quantitative ELISA, the protein is directly estimated using a set of standards and then plot a graph. In proteomics, several thousand proteins are estimated in a single run and hence it is not practical to have several standards for every individual protein.  MS technique is originally designed to be a detection methodology and not a quantitative technique.

MS is a very sensitive technique, and there is a statistical chance that certain ions are more easily picked up than others which mean that the peak height or area in a mass spectrum in itself does not accurately reflect the abundance of a peptide in the sample. The main reasons for this are the differences in ionisation efficiency and detectability of peptides. Mathematically the equation would look something like this (I will not get into the actual mathematics since that is not relevant here).

Protein concentration= MS abundance value x Error factor

The error factor depends on each run and will vary from experiment to experiment. Consider this experiment. If you have a cell lysate you run it 10 times in LC-MS/MS analysis the final result will be varied from experiment to experiment. In fact, the number of proteins identified will also significantly change and you can expect a variation of at least 30% between any two runs as shown by multiple studies. If you run 2 independent batches of LC-MS/MS for comparison then the final result will consist only of error for purposes of direct comparison. The best idea would be to compare proteins from test and control in the same run so that the error will be constant. Since the error factor is the same in both cases (which is unknown), relative fold change can be accurately calculated by comparing the abundance value of m/z peak from the experiment. 


So what is required for comparison is to run all the protein preparation that has to be compared in a single mass spec run. Now you need a method to tell which peptide came from whom. That is why we label the peptide library obtained from each case. Let us say you want to run 5 biological test cases against 5 biological control case that would be a 10 plex labelling experiment with each condition being labelled with a different label. The label will tell MS where the peptide originally came from and how much of it is there in t.

Fig 1: Hypothetical example of m/z abundance
as an indicator of fold change.
Fig 1, is a hypothetical example of m/z abundance as an indicator of fold change. Consider you are comparing 3 cases against a control sample. The height of the peak represents the peptide abundance. In comparison to control, the case 1 is slightly elevated, case 2 is drastically down and case 3 is unchanged. This kind of comparison is available for all the peptides that have been detected in MS. The overall finding is then curated by the software and presented as a protein expression data with reference to the control.

Fig 2: Labelling methods for quantification of proteins in Mass Spectrometry.
There are wide varieties of labelling methods available and different literature have a different classification and there is an overlap in some cases. For simplicity, labelling methods can be broadly classified into 3 subtypes- Metabolic, Enzymatic and chemical labelling.See Fig 2 for a summarised classification.  It is not possible to talk about all the methods and intricate details of every method, which would make this post too long. I will stick to explaining a few methods that are more famous in biological practice which will give an idea of what exactly is happening. Chemical labelling is much similar to metabolic labelling except that the label is chemically attached to a particular peptide after extraction unlike doing it metabolically. Enzymatic labelling is almost a chemical labelling except that it is done using an enzymatic process.

Stable isotope labelling by amino acids in cell culture (SILAC)

Fig 3: Example light and heavy amino acids for SILAC.
SILAC labelling was first demonstrated from Matthias Mann lab; 2002. The method is a metabolic labelling method. The core idea is that cells are given essential amino acids that carry heavy stable isotopes continuously, which gets converted into proteins in the cell. This process run for sufficient time, a great majority of the cell proteins contain heavy labelled isotopes which can be picked in the mass spec. In a typical SILAC labelling experiment, lysine and arginine residues are isotope labelled. Since trypsin digestion is commonly used for obtaining peptides this results in labelling of every peptide in the mixture. The labels are available as N terminal and C terminal labelled lysine or arginine. See Fig 3. In addition, leucine, tyrosine and methionine amino acids with incorporated isotopes have also been used as labels. SILAC method has a high efficiency but comes with inherent limitations. Other than the facts that it is time consuming and expensive it requires that the method uses a culture system only those that can be cultured are available to work with this method.

18 0 labelling


The methodology considers the idea of class-2 proteases, such as trypsin, to catalyse the exchange of two 16 O atoms for two 18 O atoms at the C-terminal carboxyl group of proteolytic peptides. Hydrolysis of a protein in H218O by a protease results in the incorporation of one 18 O atom into the carboxyl terminus of each proteolytically generated peptide. Despite its simplicity, the method is not in regular use owing to the difficulty in attaining a high labelling accuracy.

Labelling using Isobaric tags

Fig 4: Structure of TMT tags. Source
This is probably one of the most common labelling methods to be used. Let us take the example of TMT (Tandem mass tags). Labels are basically isobaric compounds (They have same net mass) with a peptide binding site.

Each chemical tag contains a different number of heavy isotopes in the mass reporter region, which gives a unique reporter mass during tandem MS/MS for sample identification and relative quantitation, a mass normaliser which adjusts for the mass and a reactive group.

I have limited the discussion on labelling methods to the basic essence to give you an idea of how the system works. I recommend you read the references to have a detailed picture of the process.

References:

Tabb et al.  Repeatability and Reproducibility in Proteomic Identifications by Liquid Chromatography-Tandem Mass Spectrometry. J Proteome Res. 2010 Feb 5; 9(2): 761. doi: 10.1021/pr9006365

Ong S, Mann M. A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC). Nature Protocols. 2007;1(6):2650-2660.

Rauniyar N, Yates J. Isobaric Labeling-Based Relative Quantification in Shotgun Proteomics. Journal of Proteome Research. 2014;13(12):5293-5309.

Sunday, July 02, 2017

Angiostrongylus cantonensis

By the end of April this year, rat lungworm or Angiostrongylus cantonensis raised an alarm with 6 cases which was apparently spreading on the Hawaiian island of Maui. The infection is called as angiostrongyliasis. it basically manifests as meningitis and if not treated can cause permanent damage to the central nervous system or death. A cantonensis is well known in South-east Asian and Pacific region and is one of the most common causes of eosinophilic meningitis. The More recently, the parasite is been found in environments of California, Alabama, Louisiana Leon and Florida regions including Alachua, Saint Johns, Orange, and Hillsborough (Where it is much more common than originally realised).

Photo 1: Adult female of A cantonensis.
Source
A cantonensis is a species related to Strongyloides stercoralis (Order strongylida, and superfamily metastrongyloidea) was first described from CSF of an eosinophilic meningitis case by Nomura and Lim in Taiwan in 1944. Morphologically, they appear like other nematodes unsegmented with a simple fully developed gastrointestinal system. Males are smaller than the females. Photo 1, shows an adult female with a dark red digestive organ, two white reproductive organs, and a transparent cuticle.

Photo 2:cantonensis egg.
Source
A cantonensis is classically an infection of rats and humans are accidental hosts. A cantonensis reside in pulmonary arteries of rats. A female lays up to 15000 eggs a day. Once eggs are laid, they hatch and the first stage larva migrates up the pharynx and are swallowed to be secreted through faeces. Snail forms an intermediary host which is infected with the larva where it develops. The 3rd stage larva in the intermediate host is the infective stage. It enters through contaminated water, ingestion of snails the larva migrates up the brain. The larvae develop into a pre adult stage and then return to the pulmonary artery, where it matures.

Fig 1: Life cycle of A cantonensis.Source
Many species of snails and slugs serve as intermediate hosts for A cantonensis. Some of the well known vectors include Achatina fulica (African land snail), Pila species, golden apple snail etc

Humans acquire the infection accidentally through ingestion of contaminated water, other infected paratenic animals (crabs, freshwater shrimps) or leafy vegetables which are not cleaned and washed well.Fig 1 from CDC page gives a detailed picture of the life cycle.

The clinical presentation will usually involve symptoms such as abdominal discomfort which progress to fever and headache. Often the case will self resolve without treatment and may not be evident. In cases of high load of parasites, the infection progresses to serious meningitis. The laboratory diagnosis is at best, a prediction. Eosinophilic meningitis and imaging studies maybe a clue. To date, there are no definitive diagnostics available. It should be noted that eosinophilic meningitis is not a definitive indicator for A cantonensis. Other agents include Angiostrongylus costaricensis, Gnathostoma spinigerum etc. Though PCR testing is available it is done only in special labs and not widely available.

In a recently published article in Digital Journal by Karen Graham, there is a discussion on what increased identification of A cantonensis means in Florida. As Heather Stockdale Warden comments, "The ability for this historically subtropical nematode to thrive in a more temperate climate is alarming. The reality is that it is probably in more countries than we found it in, and it is also probably more prevalent in the southeastern U.S. than we think".

References:

Stockdale Walden, H., Slapcinsky, J., Roff, S., Mendieta Calle, J., Diaz Goodwin, Z., Stern, J., Corlett, R., Conway, J. and McIntosh, A. (2017). Geographic distribution of Angiostrongylus cantonensis in wild rats (Rattus rattus) and terrestrial snails in Florida, USA. PLOS ONE, 12(5), p.e0177910.


Wednesday, June 07, 2017

Superdrug for the Superbug

Antibiotic resistance is a huge problem and the problem of so-called "Superbugs" is not new to the readers. There are several reports of resistance against drugs that are considered as reserved only as last resort. Colistin is an example, which is preferentially used against gram negatives as the last drug of choice. Similarly, Vancomycin is a reserved antibiotic for gram positives, especially MRSA. MRSA now being an absolutely common isolate vancomycin is more commonly used.

Fig 1: Mechanism of Vancomycin resistance. Source
Vancomycin (Initially code-named as 05865 and later marketed as Vancocin) was first isolated by Edmund Kornfeld in 1953 from a soil sample collected from Amycolatopsis orientalis from the interior jungles of Borneo. The toxicity was a limiting factor and antibiotic was reserved only for those cases, where nothing else worked. Chemically, Vancomycin is a glycopeptide (branched tricyclic glycosylated nonribosomal peptide) and acts by inhibiting cell wall synthesis. Vancomycin binds to the terminal D-Ala-D-Ala dipeptide which halts the process of peptidoglycan formation. The resistance is due to change in D-Ala-D-Ala. The most common change is D-Ala-D-Lac which has orders of low binding of vancomycin. See Fig 1, for details.

Vancomycin resistance has been a huge problem in health care settings. There are several genes that mediate resistance to vancomycin named as VanA, VanB, VanC, VanD and VanE. The commonly talked about vancomycin resistant strains are VRSA (Vancomycin resistant Staphylococcus aureus) and VRE (vancomycin resistant Enterococcus species). These are very difficult to treat infections. The most common mechanism of glycopeptide resistance is to develop modified cell membrane receptors with reduced affinity. The problem has been recognised quite early and one of the obvious methods of dealing with it is to modify the vancomycin. As early as 1999, a collaborative group between Princeton and Merck showed that the carbohydrate derivatives of vancomycin were able to overcome resistance to vancomycin.

Fig 2: Structure of[Ψ[C(═NH)NH]Tpg4]vancomycin
aglycon. Source
There are several reports in the literature reporting successful modifications of vancomycin increasing its kill capabilities. Many of these were loosely called as Vancomycin ver 2.0. In 2011, Dale Bogger's lab from Scripps Institute reported the development of a new vancomycin derivative a [Ψ[C(═NH)NH]Tpg4] vancomycin aglycon which could bind both d-Ala-d-Ala and d-Ala-d-Lac. 

Fig 3: Structure of modified vancomyin 3.0. Source
Improving on their work Bogger's lab has now reported the design of a Vancomycin 3.0 (CBP  C1- aminomethylene vancomycin). This chemical has 3 different modes of activity. Not only it has a dual binding activity, the addition of quaternary ammonium salt in the structure (It was a calculated design), provided a binding pocket-modified vancomycin analogue which was independent of D-Ala-D-Ala or D-Ala-D-Lac binding. Another modification of peripheral (4-chlorobiphenyl) methyl (CBP) to the vancomycin disaccharide made it highly potent. The same drug also can induce cell permeability. Basically, the same drug has 3 independent mechanisms of action, making it difficult for the bacteria to acquire resistance. Indeed, there is data to show that in vitro the bacteria didn't acquire any resistance even after 50 passages.

As Bogger States, "Organisms just can't simultaneously work to find a way around three independent mechanisms of action. Even if they found a solution to one of those, the organisms would still be killed by the other two".

The compound is not yet ready for human clinical use. The chemical synthesis is very laborious and it is not known as to what are its side effects. 

References:

Levine D. Vancomycin: A History. Clinical Infectious Diseases. 2006;42(Supplement 1): S5-S12. 

Ge M. Vancomycin Derivatives That Inhibit Peptidoglycan Biosynthesis Without Binding D-Ala-D-Ala. Science. 1999;284(5413):507-511.

Xie J, Pierce J, James R, Okano A, Boger D. A Redesigned Vancomycin Engineered for Dual d-Ala-d-Ala and d-Ala-d-Lac Binding Exhibits Potent Antimicrobial Activity Against Vancomycin-Resistant Bacteria. Journal of the American Chemical Society. 2011;133(35):13946-13949.

Okano A, Isley N, Boger D. Peripheral modifications of [Ψ[CH 2 NH]Tpg 4 ]vancomycin with added synergistic mechanisms of action provide durable and potent antibiotics. PNAS. 2017;:201704125.