Tag: Immunology

“PREP-TO Qualify” – Tips for Qualification/ Re-class examination

by Alphonse Lab, posted in Education, General, Non-specific, Uncategorized

Prelude

Ma was elated as he looked at the temple from a distance.

As he came close, the edges of the stone stairs welcomed him back into his familiar world.

“These stones are surprisingly uniform”, thought Ma as he took the steps back into the temple.

“Am I learning anything in these journeys?”

Ah! Here is the world I learn a lot from! thought Ma

“What did you learn in this trip?” asked the Zen Master.

Ma looked puzzled and couldn’t hide his frustrations.

“Why do I have to do this?”

“Why do you make me do it every year?”

“You make me travel to my village. I walk the same road, cross the same fields and valleys. I travel the same bridges every year. I anticipate change and gape at even the subtle ones. Without a thought I wave at the children who happily cross by.

Familiarity is breeding boredom, so I am lost in my thoughts! I start to think what would have changed in my village, who would have ceased to dream or even worst whom I know would have ceased to breathe. This is a journey I forebode every year and return back thinking about all this in reverse all over again.”

“What do you expect me to learn? Don’t I learn what I should here in this sacred temple?” blurted out Ma

The journey to Qualify

Do we not learn what we should by doing research? Don’t we all learn in the space between lab and desk? Why should we have to go through this so called “Qualification or reclassification” in order to get a PhD? Didn’t we get admission through selection? Why is that not enough to qualify?

As we prepare for our qualification exam, probably these are some of the question that go through (most of) our minds.

Well, everyone of us know the answer to these questions if we give into the process. I guess, it is within each individual to perceive the potential as we see fit!

Qualification exam is a journey, a passage that we take in the process of getting a PhD. As we prepare to take this exam, the preparation itself becomes a journey and the exam is an experience that aims to mold us as “independent researchers”. “The more we are prepared for this journey the better and richer our experience”.

This article aims to be an aid/tool in this preparation process. It is more general and comes out of my experience. I would urge you to go through this tool and take away things that may resonate with you. Take things that you think may help you. Leave the others. Your journey is unique and if this blog helps, it has met its purpose.

I would like to encompass the complete preparation as

“PREP TO Qualify”

P-R-E-P (Proposal, Review, Examiners, Practice)

DSC_0012_edit_toned_focusProposal:

Proposal is an important document (to describe the project) to the examiners and a lot will depend on its preparation. Nothing new, I know! but it is important to keep in mind as we prepare the document, that this document will give away the plan for the next couple of years and how well you are prepared for the journey.

It is no secret, but a gentle reminder that, “a well defined project will define your qualifying exam”.

  1. Follow instructions:
    Stick to rules, prepare as per required format, do not go overboard.
  1. Preliminary data helps, definitely helps!!
  2. Ask yourself! Does the proposal have the potential to say a story? A story is better received!
  3. Take care in designing the experiments. Know the time it will take to perform and complete the experiments. It is important!
  4. Take the comments from committee members seriously as you prepare the document.

Review:

Review the “area of research” and the subject background. It is what you are tested on! It is important to prepare well by reviewing the material at hand.

  1. Start early to prepare
  2. “Plan your preparation”. Have a strategy to cover extensively the research area and subject background.
  3. Take time to read previous work done in the lab (related and unrelated)
  4. Give enough time for subject background (specialization area)
  5. Cover recent development in the field of research.
  6. Don’t forget to learn about the recent developments in experimental techniques pertaining to the field

Examiners:

  1. Know your examiners.
  2. Learn about their research and techniques in their lab
  3. If they take any courses in the area of your research. Cover the bases
  4. Their focus on research (strengths) and interests.

It is easy to ignore this, but don’t.

Practice:

  1. Give your proposal to peers in the lab. Get their feedback. A good peer will not be judgmental. It is better to “fine tune” your proposal before submitting to the examiners. It never hurts in getting some advice.
  2. Welcome questions from your peers from the lab or outside. Field their questions from the subject and project. It will definitely help on the day of exam.
  3. Practice fielding hypothetical questions, including experimental design ones!
  4. Practice your presentation talk in advance.
  5. Revisit your plan at regular intervals and re-strategize your preparation accordingly.

With PREP under control, it is before the exam and on the day that you have “T-O” (Taking control, On that day) qualify.

Taking Control:

  1. Reduce your stress before your exam
  2. Plan for the day
  3. Practice your talk prior and be comfortable with the slides.
  4. Keep it simple. Remember, the examiners have already read the proposal. So go easy with the presentation.
  5. Remember to relax. Read non subject articles or books.

On the day:

  1. Eat well on the day of exam.
  2. Dress comfortably and know that you are prepared and planned for this day.
  3. Keep a bottle of water during your exam.
  4. Take time (if you need) to answer the questions.
  5. Remember, it is alright to say “I don’t know”. It is better to say “I don’t know” than half guess the answer, because it will avoid further questions from that half guess.
  6. Be prepared to draw figures and be willing to use the chalk board to explain (You may be made to draw on purpose!).
  7. It may be hard, but try to enjoy the process of the exam. After all, you are the expert fielding the questions!

All the best to all who PREP-TO Qualify!

Epilogue:

After hearing patiently Ma’s frustration, the Zen Master turned back to retire to his room.

“Don’t you want to know what I learnt?” asked Ma

“I have!” said the Master

-Author: Martin P. Alphonse

Epigenetics “train” innate immune memory!

by Alphonse Lab, posted in Specific-Innate Immunity
Prologue

The mound beside the temple graced his seat. And the evening lights of the village from a vantage point set the scene for Zen Master’s daily contemplation.

Ma cautiously approached and sat beside him.

The dim evening lights of the village invited their thoughts and minds.

“How was your day?” asked Zen Master

Trying to recollect his memories,

Ma replied with a smile

“You train me well.”

And after a pause, he continued

“I will remember everything you teach me and one day I will become a Zen Master just like you”

“Can you remember everything?” asked Zen Master realizing the importance of his training

Ma tried to understand the tenor of the question in his mind.

“Can I?” inquired Ma with hesitation, quickly giving up his thoughts.

Immunological memory

Immunology memoryMemory in our immune system is indispensable. Immunological memory is the ability of the immune system to respond more rapidly and efficiently to pathogens that have been previously exposed. Threat from invading pathogens and environmental triggers (henceforth antigens) are constant to the host. And to cope with these threats, immunological memory is pivotal in its recognition and elimination.

Based on the ability to respond to antigenic challenge and contingent on

  1. Specificity and
  2. Clonal selection (theory), which leads to Immunological memory, the immune system is classified into innate and adaptive immunity.

When encountered with an antigenic challenge, adaptive immune cells such as T or B lymphocytes adapt (hence the name adaptive) in response to the antigen. This response is specific and as one of the outcome, memory population (to that specific antigen) are generated. And when re-challenged, the memory response is rapid and robust.

However, innate immune cells such as granulocytes, macrophages, monocytes and Natural Killer (NK) cells were thought to be completely non-specific and lacked memory response.

Identification of pathogen pattern recognition (PPR) receptors in innate immune cells has reasoned the complete non-specificity dogma. It is now appreciated that innate immune system has a semi-specific recognition of pathogen associated molecular patterns (PAMPS) from different classes of microbial pathogens. And now, experimental studies and proposed concepts stack up in support of innate immune memory.

Innate immunologists argue that epigenetics is the key that unlocks the memory of innate immune system!

Epigenetics ”train(ed) innate immunity”:

Epigenetics focuses on cellular and physiological phenotypic changes that are caused by external or environmental factors. In essence, these factors turn on or off the gene expression and affect how the cells read genes instead of being caused by changes in DNA sequence.

When innate immune cells (such as macrophages) are treated with fungal structure β-glucan, positive epigenetic modifications occur in histone regulatory proteins (H3K4me1, H3Kme3 and H3K27ac). These changes are responsible for increased response with gene expression of host defense molecules in innate immune cells when re-stimulated with same or similar stimuli. Innate Immunologists argue that, epigenetic alterations due to exposure of pathogens in innate immune cells in vertebrates can equip them with an “antigen independent memory”. So when re-exposed to same or similar pathogens, there is heightened response. They propose such memory as “trained immunity”.

They insist that epigenetics in innate immune cells leading to memory response makes sense at an evolutionary stand point. Evolutionary conservation theory of “systemic acquired resistance” is proposed in innate immunity. This stems from the fact that innate immune memory is seen as host defense feature in organisms such as plants and invertebrates that lack adaptive immunity (Conventional adaptive immune memory is present only in vertebrates).

Furthermore, role of epigenetic modification in innate immune memory have also been shown in NK cells. Studies have shown that these ‘memory’ NK cells can rapidly degranulate and produce inflammatory cytokines upon reactivation.

It is proposed that these epigenetic modifications of cellular function is at the core of long term training of immunity for innate immune cells.

Other proposed mechanisms that are thought to play a role in innate immune memory include Long-term regulation of Non-Coding Ribo-Nucleic Acids (lncRNA) and metabolic pathways including glycolysis and Warburg metabolism are implicated.

Is memory forever?

Given the specificity and gene rearrangement with central and effector memory cells, adaptive immunity is for lifetime.

How long does the ”trained” innate immune cells have memory? Whether the altered epigenetics is retained after proliferation? Can it be transmitted epigenetically in the germ line (as seen in plants)? are important outstanding questions that remain to be answered.

In summary, classical adaptive immune memory is specific, antigen dependent and is mediated by gene rearrangements. Innate immune cells were thought to be completely non specific which lacked memory response. However, recent studies show that innate immune cells can be trained to acquire memory. Trained innate immune memory is semi-specific, antigen independent and can be mediated through epigenetic reprogramming.

Epilogue

Realizing the confusion in Ma’s voice,

Zen Master filled the silence with his consolations

“Train your mind! Be open to all the environment has to offer. Absorb the harmonious changes and try to preserve the experience in your memory. Your trained mind will adapt as need arises!”

“But, will the environment make me a Zen Master like you?” asked Ma

Waiting for an affirmation from his adoring master, his wanting eyes gazed at Zen Master

His glance spoke thousand words to Ma and he returned his gracious

Smile!

References:

  1. Murphy, K. M. (2012). Janeway’s Immunobiology (pp. 1–892). Garland Science.
  2. Netea, M. G., Latz, E., Mills, K. H. G., & O’Neill, L. A. J. (2015). Innate immune memory: a paradigm shift in understanding host defense. Nature Publishing Group, 16(7), 675–679. http://doi.org/10.1038/ni.3178
Nano-Immuno-technology

The dawn of Nano-immuno-technology: Applications in Vaccinology

by Alphonse Lab, posted in Specific-Immunotechnology

Prologue

The morning was about to dawn and the radiance of the golden hour filled the sky. Swayed by his overwhelming senses, Ma was standing against the waves of the ocean caressing his feet and its sounds permeating his ears. As he turned around, he saw Zen Master walking towards him

“What did you learn?” he asked

“The massive ocean is so overwhelming, I don’t know where to focus!” replied Ma with concern

Understanding the impatience in Ma’s voice, Zen Master with all his mature wisdom, whispered:

“Search! Slowly try to focus on what interests you. The key is finding your interest. Look around, things you see and the one’s you don’t. Stay with it! Make that interesting point your passion. With time, the passion will become so communicable that you will steadily experience the overwhelming ocean with your mind. And in that oneness, pay attention; the ocean will teach you what you have to learn. Remember, the key is finding your interest!” 

The meeting point of Nantechnology and Immunology

Oceans they are! The discipline of Nanotechnology and Immunology are boundless. Trying to find an interesting point of focus at their confluence is as intriguing as the subject themselves. Therefore, the aim of this short digest is not to cover all the important advancements or possible applications; but rather an attempt to focus at the accent point of the fascinating intersection of both worlds.

Nano-Immuno-technologyA nanometer (nm) is a unit of measurement that equals to one billionth of a meter. The technology that uses the unique properties of objects within the overall size range of 1-1000 nm is called Nanotechnology. Yes, size does matter! Especially when it comes to immune responses. Bio substances that induce an immune reaction, including antigens or pathogen associated molecular patterns (PAMPS) are in nm scale. The physical and chemical properties of a substance such as size, shape, and hydrophobicity determines its ability to induce an immune response. And the ability to engineer properties of substances towards modulating an immune reaction, fields the application of nanotechnology in Immunology. And what an interesting field it is! The last decade or so has seen an exponential advancement in the field of nanotechnology. Limited by scope, this short summary aims to focus on its application in development of vaccines.

Road to Vaccinology via Nanotechnology

Nanoparticles as adjuvants

The goal of an effective vaccine is to enhance the quality and quantity of the adaptive immune response (both the humoral and cellular). To this effect adjuvants like alum are commonplace in vaccines. An adjuvant is a substance that is added to a vaccine to increase the body’s immune response to the vaccine. Nanoparticles in form of nanoemulsions (oil-in-water emulsions) that are composed of solvents and surfactants provide such adjuvant activity. An example in current use is MF59 (Novartis), which consists of squalene oil (a natural 30 carbon organic compound from shark liver oil or amarnath seeds) in combination with polysorbate 80 and sorbitan trioleate. MF59 is licensed as adjuvant in influenza virus vaccines in Europe under the commercial name FLUAD®. MF59 have been shown to be a more potent adjuvant than alum for inducing humoral and Th1 type cell mediated (cellular) immune response. It is interesting to note that MF59 is yet to be approved in the Americas. Apart from nanoemulsions, nanoparticle based vaccine carriers that are in development as adjuvants include biodegradable poly(lactide-co-glycolide) (PLGA) nanoparticles, co-polymer hygdrogels or ‘nanogels’, cholesterol-bearing hydrophobized pullulan (CHP) and cationic lipsosomes. These nanoparticles raise interest in the field due to their size and immunomodulation properties.

Novel vaccine strategies

Nanoparticles as adjuvants may sound unimaginative. Imagine this! How about engineering nanoparticles as complete vaccines? That should sound novel in approach. Right? Well, such vaccines are in development and in preclinical and close to clinical trials. Although some genuine ethical concerns do swell against these vaccines, the immunological outcome looks promising. Virus like Particles (VLPs) and Self Assembling Protein Nanoparticles (SAPNs) are a two such nanoparticles engineered towards development as complete vaccines.

Virus like Particles (VLPs) based vaccines

With their ability to replicate and undergo genetic recombination, viruses are the smallest among pathogens and yet ingenious ones that find ways to evade our defense mechanisms. Taking cues from viruses, Nanotechnologists have developed virus like particles (VLPs) as novel strategies for vaccines. VLPs unique size, shape, uniformity and stability of structures closely resemble viruses. However, they do not contain genetic material and lack replicative abilities. VLPs are nanoparticles (20-100 nm) that can be broadly divided into two categories, Non-synthetic VLPs and Synthetic VLPs.

Non-synthetic VLPs can be designed to contain viral protein subunits that have the ability to form a viral capsid. Currently, there are about 20-30 different enveloped or non-enveloped VLPs in preclinical and clinical development against infectious diseases and cancer. As a strategy, one of the main advantages of VLPs that could be exploited is incorporation of other biological structures such as Toll Like Receptor (TLR) ligands, cell targeting moieties or other biologically active mediators that could enhance the efficiency of vaccines.

Synthetic VLPs are derived by chemical synthesis of lipopeptide monomers that could enhance nanoparticle assembly and stabilize the three-dimensional conformational structure of protein antigens. One promising example of Synthetic VLPs that is currently in development is Lipopeptide-based synthetic VLPs (20-30 nm), that have been used to repetitively display a peptide-mimetic epitope derived from V3 variable loop of gp120 protein of HIV virus.

Self Assembling Protein Nanoparticles (SAPNs)

 Learning from microbial pathogens and VLPs, SAPNs are designed using systemic modeling. By taking advantage of both naturally occurring and synthetically engineered biomolecules, SAPNs are designed to optimize immune responses to vaccine antigens. In essence we could call SAPNs as Version.2 of VLPs. SAPNs can be designed to achieve repetitive antigen display for effective vaccine development. In the process, it can enable conformational presentation of inserted protein epitopes in exposed configuration that could protrude or extend from the surface of the particle after assembly of subunits. About 180 peptide chains can be assembled into a single nanoparticle to form an icosahedral structure. The advantage in this geometry enables better antigen display and cellular activation that could lead to significant increases in the specific production of high-titer, high affinity antibodies directed against the inserted antigen epitopes. A successful example of SAPNs is the incorporation of trimeric coiled-coil epitopes from surface protein of severe acute respiratory syndrome coronavirus (SARS-CoV). These SAPNs has been shown to strongly induce virus-specific neutralizing antibodies in mice after immunization. Similar SAPNs specific to malarial parasite Plasmodium falciparum and P. berghei are in development.

Immunologists consider vaccines as an integral part in prevention and fight aginst infectious diseases. Apart from infectious diseases, advantages of vaccines have found ground in fight against chronic diseases and cancer. At this juncture, application of nanotechnology in form of adjuvants and novel vaccine (VLPs or SAPNs) strategies buoy up the world of vaccinology. Over the long haul, some of the ethical concerns regarding the use of nanoparticles are valid and inflammatory status and long-term effects of these nanoparticles should be extensively studied. Yet, important developments in the field of nanotechnology have already found its application in fields of immunosuppression, transplantation and cancer immunotherapy. Furthermore, exploiting its use in experimental immunology both in vitro and in vivo can provide novel application methods and can potentiate our understanding of our immune system.

Epilogue

 The rays of the morning sun warmed Ma, as he was charged with vim and vigour from Zen Master’s counsel.

Little did he know that he had already stumbled upon his point of interest. I should tell this to the Zen Master he thought, but with a smile returned home thinking to himself

“Tomorrow is another day, yet another morning will dawn to teach more. The ocean is full of lessons!”

References:

(1) Smith DM, Simon JK, Baker JR. Applications of nanotechnology for immunology. Nat Rev Immunol. 2013 Jul 25;13(8):592–605.

(2) Torres Andón F, Alonso MJ. Nanomedicine and cancer immunotherapy – targeting immunosuppressive cells. J Drug Target. Informa Healthcare; 2015 Aug;23(7-8):656–71.

(3) Abuelma’atti MT. Nanotechnolgy: Benefits, risks and ethical issues. Exhibition,    “Innovative Engineering for Sustainable Environment.” IEEE; 2009. 5 p.