Monthly Archives: December 2013

Using Antibodies as vaccine delivery vehicles

Antibodies are glycoprotein molecules synthesized by plasma cells (mature, activated B cells) with the capacity of binding to any potential antigen epitope. For a review of lymphocytes and how they are activated, see this link where you will find more information about antibody production in response to ’challenge’.


An (IgG) antibody with basic structural features labeled

Antibodies are the natural products of these plasma cells and function in a variety of ways to effect immunity. Most basically, they bind and may interrupt the function of the target molecules or trigger a response disadvantageous to the pathogen. In addition, a number of other functions are mediated by these molecules, including recruitment of complement and of phagocytic cells that will digest and inactivate the cell / antigen.

Therapies, such as vaccines, are designed to separate and eliminate the disease-causing elements of a pathogen from those that generate an immune response, thereby initiating a normal immune response to antigens without the dangerous exposure to live pathogens. Most often, these are prophylactic vaccines that initiate the development of immune ’memory’ prior to any disease exposure.

In some cases, therapeutic vaccines do much the same job, but are used to ’jump-start’ an immune response that has failed to initiate naturally for some reason (this may be because the target of the therapy is very similar to ’self’ as is the case with cancer), or because a long-term, chronic disease has fooled the body into tolerating an unwanted condition.

Additionally, some molecular therapies provide passive immunity by administering exogenous antibody that fulfills these functions. A weakness of these therapies is that, by providing pre-made antibody, potential antigens are blocked and no endogenous antibody response will be elicited.

A final use of antibodies, to be elaborated further here, is to provide targeted delivery of toxins to pathogens or infected cells or to deliver antigens to the immune system.

Purpose: to trigger / amplify immunity to an ongoing infection or disease


1. Target protein or cell – what cell and what protein on that cell should be targeted to elicit the desired immune response?

2. How to get antibody to the site where target cells are present?

3. What is the desired response / activity of the target cell?

4. What, if any, molecule is being delivered to these cells?

5. Lastly, how can efficacy be measured and what are the objective endpoints that will be used to determine whether therapy is effective?

Although this antibody is not currently in use therapeutically, I will use, as an example, one that I made while working for a biotech company some years ago.


An antibody with an antigen conjugated to the Fc portion

The antibody we used specifically bound to the macrophage mannose receptor (MMR) expressed by macrophages and the similar phagocyte cells, dendritic cells. Natively, this protein binds to a sugar, mannose, that is commonly charged to protein molecules. Once bound, the MMR will direct receptor-mediated endocytosis of the bound protein and deliver it to endolysosomes for processing and presentation upon MHC class II molecules (see animation below). As explained in the link, processing and presentation lead to the activation of T Cells and the resulting immune response.

Using an antibody that targets this molecule (MMR), a target compound can be fused to the antibody (chemically or genetically) leading to the precise delivery of this compound into the cell and the generation of a response. The antibody will guide the (tumor) antigen to the phagocytic cell. In this way, the antibody serves only as a vehicle. This vehicle takes its passenger, the antigen that we would like to generate an immune response against, and inserts this antigen into the processing and presenting apparatus of these ‘professional’ antigen presenting cells.

Animation of Antibody delivering a Target Antigen to an APC:

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Posted by on December 26, 2013 in Uncategorized


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reviewing TEAS test questions

In preparing for a new semester, I’ve been downloading a few free question compilations for the TEAS test. TEAS is the ‘Test of Essential Academic Skills’ that students wishing to enter into nursing programs must take. Since many of my students in both my Biology and Microbiology classes wish to enter nursing programs and are taking required classes for that purpose, I find these questions to be very useful in directing my syllabus creation.

However, today, I found this question amongst the free samples offered at the website:

Category – Science

Q – What is a term used to describe the subtle energy running through the body?
A. The Tao
B. The Force
C. Chi
Your answer: –
Answer: C – Chi is a term used for the subtle energy that runs through the body.
Chi is specific to Traditional Chinese Medicine. Other terms used are ki, prana and life force.

What am I to think?

I’m not sure that I am comfortable with ‘Chi’ being a legitimate concept being taught in science classes.

Am I wrong to think that the science and medicine taught to students of these disciplines should be objectively verifiable? Is ‘Chi’ a documented phenomenon? Or is this just a word / description that describes some metaphysical concept?

Wikipedia uses the following to define Chi:

Qi is the central underlying principle in traditional Chinese medicine and martial arts…Elements of the qi concept can also be found in Western popular culture, for example “The Force” in Star Wars.”

Given this loose definition for an equally loose concept, I think “The Force” is an equally reasonable answer to the question as it is posed. And no answer to this question should be acceptable or expected in a serious exam. I certainly hope that this question is an example made up by, and not one that my students are ever likely to see on a real examination.

See this link for a review of chi, also known as qi or qidong, in medicine.

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Posted by on December 26, 2013 in Uncategorized


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One day, two bad movies

ImageCinematic masterpiece #1: Videodrome, staring James Wood playing a human sized vagina used to store videotapes and small firearms and a script Samuell Beckett would throw away for being senseless.

Videodrome doesn’t deserve and stars at all.


Opus #2: Hard to Kill starring Steven Segal, Kelly LeBrock and a mystery that makes Blue’s Clues look like it was conceived from the mind of Dr. Moriarty. “I’ve tried analyzing the tapes for anything … common phrases … like the Senator’s ‘You can take that to the bank’ that he keeps repeating in his commercials and also clearly said in your secret tape.” Segal also makes martial arts look like the province of any overweight couch potato. Breaking arms, legs, fingers and necks takes about as much strength in his hands as snapping a twig.

Hard to Kill at least is somewhat enjoyable if you like this sort of thing.


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Posted by on December 21, 2013 in Uncategorized


One base at a time…

In 1977 Fred Sanger’s lab developed a method for determining the DNA sequence of short fragments. I touched on this briefly in a prior post published here at the time of Dr. Sanger’s death.

Over the subsequent decades the technique was refined and eventually transformed into a single-tube automated reaction, however the basic method remains the same. There are three basic principles that underlie the Sanger dideoxy DNA sequencing method.

The first principle is that DNA is sequencing is a modified replication reaction that occurs whenever a cell divides. This is accomplished by stringing nucleotides together according to the original DNA molecule used as a template.  For a brief review of this replication reaction, see the animation below from HHMI.

The second principle is gel electrophoresis, the use of acrylamide gels to separate DNA strands based on their length. Acrylamide forms a weblike polymer sieve through which molecules (like DNA) can move. Because larger molecules get hung up on the threads of this web more often than smaller molecules do, the larger ones cover less distance in the same amount of time. Also, because DNA has a uniform negative charge spread out along its length, when an electrical current is run through the gel, the DNA will migrate toward the positive pole. If the acrylamide is made at just the right density, the DNA fragments can be separated to such precision that single base differences in length are distinguishable.

In the animation below, four tubes are prepared, each with fragments of one size. These are loaded into ‘wells’ in an acrylamide gel and then subjected to an electrical current.

The second principle comes from the nature of DNA itself and the chemistry of the nucleotides that make it up.

DNA is a long polymer made up of many nucleotides. The name, DNA, stands for deoxyribose nucleic acid, which describes the molecule chemically. The prefix ‘de-‘ means that DNA nucleotides lacks something that standard ribonucleic acids have. The ‘oxy’ part tells us what is missing, an hydroxyl (-OH) group. (See figure below) The first hydroxyl group is the one that determines the difference between DNA and RNA.


A ’di-deoxy’ molecule lacks an additional hydroxyl group (dideoxy= two hydroxyls missing)


This second hydroxyl is removed from a position that forms the backbone of the molecule and is required for the next nucleotide to attach in a polymerization reaction. Without this, DNA replication comes to a screeching halt. If a sequencing reaction, which is a form of a polymerization reaction, includes a portion of these dideoxynucleotides, then the incorporation of this nucleotide will terminate the reaction at a known base.

Because DNA is comprised of the four bases, (A)denine, (T)hymine, (C)ytosine and (G)uanine, deoxynucleotides with each of these four bases are required for DNA synthesis. If a synthesis reaction is supplied all four of these in amply supply, then synthesis will proceed smoothly. If one of these is omitted and replaced with only the dideoxynucleotide version, then synthesis will proceed until that dideoxynucleotide is incorporated. Because this nucleotide lacks the hydroxyl group required to attach a subsequent nucleotide, the reaction stops.

This doesn’t give us much information, however, because we can only read up to the first of each type (A,T,C or G). What is done then, is that all four deoxynucleotides are supplied, but in each of four tubes, a small proportion of dideoxynucleotides is added. In this way, the synthesis reactions can proceed until a dideoxynucleotide is added, but this may happen at a different occurrence of this nucleotide in each instance of synthesis.

Consider the template sequence below in black. Replicative strands are made using deoxyribonucleotides (in black) and dideoxy-A (in red).







When these fragments are run on a gel, we can visualize a band at positions corresponding to the occurrence of each ‘A’ nucleotide in the sequence.

In the same way, three additional reactions are run including dideoxynucleotides of each flavor and then run on separate lanes of the gel. Altogether, these four lanes provide a complete account of the original DNA sequence.

full gel

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Posted by on December 18, 2013 in Uncategorized


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A Book and an App


Bring just one pencil

The Book

I’m enjoying a new (to me) book over the holiday break: The Mysterious Benedict Society, about several children- but one in particular – who are recruited by the odd, reclusive, Mr. Benedict. Their recruitment, itself, is a bit of an adventure. Each responded to this strange add appearing in the newspaper.

There are tests within tests to separate the wheat from the chaff. A test that’s a puzzle, a maze, tests of resourcefulness and honesty. And, when all that is over, the real work is just begun.

I originally bought this book for my son, who read a few pages and then decided that there wasn’t enough action in it and set it aside. But, seriously,  buying for Harry is just cover for me to get whatever I want regardless of the age of the target audience, so I wanted to read it from the start.

I’ve heard of people doing book clubs specializing in just children’s books and it’s no wonder. The youth – young adult book market has exploded over the past decade or more as every author vies to be the next JK Rowling. Sure, it’s put a lot of crap into circulation, but there are also a lot of extraordinarily creative authors getting published who may not have seemed worth the risk fifteen years ago.

I’ve only just started the Benedict Society this weekend, so I can hardly give a fair review, but as far as I’ve read, I’m enraptured and can’t help but to want to spend my days lying in front of the fire reading.

The App


Big Trak

The app I found today is called ‘Cargo Bot.’ It’s a puzzle game that introduces kids to programming algorithms in a way that they can immediately see working and grasp the concepts. I recommend it for any child (or even adult) interested in learning how computers think. It’s a little reminiscent of the late 70s programmable tank toy, Big Trak, except this app is actually fun for more than two minutes.

Imagine all the fun you can have delivering apples to your dad with your own Big Trak and transported (sold separately)! I thought this thing was the bee’s knees back then, but didn’t ever get my hands on one( it sold for a whopping $43) until much later when my friend Kevin and I were talking and he mentioned that he still had one.

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Posted by on December 14, 2013 in Uncategorized


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Another Semester Ends .. A quick note

Suddenly, the end of every semester rushes in surprising me with how little time is left to finish the material I wanted to cover. As with any semester, I could have used more time. Despite reaching the end of my syllabi, I could easily have used another week or more to fully cover that material for each class.

But done is done. All in all, I’d call it a good semester.

But what’s next?

My next semester brings a return of Microbiology, where I will again try to sit material from the front end of the course in order to have more time to cover immunology at the end.

I will also be teaching Ecology. This will be a first time for me doing this class, so I expect it to be interesting. My ideal is a discussion-based class where we do a bunch of reading, maybe watch a film or two and then talk through the big ideas. Because I expect us to spend a good amount of time talking about evolution, we will be reading Jerry Coyne’s Why Evolution is True, and follow his blog for some spontaneous talk about evolution and related topics (like cats).


HHMI’s Earth Viewer complements readings from Shubbin’s Your Inner Fish

In the meantime, I’ve been invited to speak on the use of technology in the classroom- featuring my use of the iPad to present an interactive handbook, apps that go along with course material, games (and/ or gamification) as learning tools and other online resources like HHMI, or iTunes Course Manager.

I look forward to the opportunity to find out what others are doing in this area and maybe even find collaborators to help put together even better materials.

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Posted by on December 13, 2013 in Uncategorized


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Mom always says …

Don’t Play Ball in the House.

It could happen to you.

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Posted by on December 9, 2013 in Uncategorized


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Antigen Presentation #3: MHC Class I

Antigen Presentation

Presentation by Epithelial Cells

Consider: Under what circumstances would any cell in the body need to initiate an immune response?

Here, I’m using epithelial cells as an example, however, every cell in the body has the ability to present antigens on MHC Class I. In fact, it is a normal, continuous process that the cells cannot fail in without consequence.

While professional APCs process and present antigens that they have phagocytized, other cells divert a small amount of the total protein they make towards MHC I presentation. This allows the immune system to constantly observe these cells and ensure that they are not suffering gross mutations or infections. Most of the time, cells produce normal, ‘self’ proteins against which there are no T Cells (due to negative selection – see Lymphocyte Development).

In the event that MHC I expression is subverted, these cells are presumed to be infected and will be targeted for killing by special cells called Natural Killers, or NK Cells.

If a Non-Self antigen is presented by MHC I, these are recognized by CD8 T Cells. Like the reactions between APCs and CD4 T Cells, cells expressing MHC I + Non-Self Ag engage T Cells via their unique TCRs. The only difference is that these T Cell : Presenting-cell complexes are stabilized by CD8 molecules on CD8+ Killer T Cells.

ImageThe result of this binding is the activation and proliferation of Killer T Cells that will turn upon and kill the presenting cells by releasing perforin and granzymes that perforate target cells and trigger apoptosis (cellular suicide).

Keep in Mind the Big Picture!

To summarize with an example:

  1. Host cells are infected with a virus
  2. The virus replicates within the host cell, producing viral proteins in the process
  3. Some of these proteins are diverted to proteases that digest them and load the antigen fragments onto MHC I molecules
  4. The MHC I +Ag is transported to the plasma membrane to ‘present’ Ag
  5. MHC I +Ag is recognized by a T Cell bearing an TCR specific for the MHC+Ag complex. This interaction is stabilized by CD8 binding to MHC I.
  6. If a stable interaction is formed, the T Cell will become activated, meaning it will proliferate and secrete perforin and granzyme toward the presenting cell
  7. Perforin and Granzyme will lead to the apoptotic death of the presenting cell
  8. By killing the infected cells, the infection can be stopped before spreading farther in the body.

A CD8 T Cell (the smaller cell) killing a virally infected host cell:


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Posted by on December 8, 2013 in Uncategorized


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Antigen Presentation #2: B Cells

Antigen Presentation

Presentation by B Cells

Before thinking about B Cells presenting antigen, first recall that B Cells are lymphocytes bearing antigen receptors on their surface called B Cell Receptors (BCRs). These BCRs have been randomized during development such that every B Cell can theoretically bind a unique antigen. See Lymphocyte Development for a refresher on this if you need it.  The major function of B Cells is to make antibody that is nearly identical to its receptor protein, which will be secreted and can then bind to antigens of the same shape.


B Cell with specific BCR engages an antigen on a bacterium (Left). After activation this B Cell will become a Plasma Cell secreting antibody with identical specificity as the original BCR (Right).

A major distinction between B Cell phagocytosis and that by Macrophages is that B Cells only take up materials they have bound with their BCRs, while macrophages take up material indiscriminately. The reason for this, of course, is that B Cells are gearing up to produce antibody, and the best way to ensure this antibody will bind anything of use is if only B Cells bearing specific BCRs known to bind antigen are activated. Macrophages have no antigen-specific receptors, so this specificity is not required by those cells. The membrane bound BCR is exactly the same molecule as secreted antibody – except for the small portion that anchors the BCR to the membrane.

Like macrophages, B cells are ‘professional’ antigen presenting cells (APCs) that take up exogenous antigen, break it down within lysosomes and present the resulting peptide fragments within MHC Class II Molecules. As with other professional APCs, this is intended to pick up foreign, invasive particles for present them to T cells to elicit a specific immune response.

ImageJust by binding to antigen with their BCRs, the B Cell will become (at least partially) activated, stimulating proliferation of this cell and processing/presentation of antigen as indicated above. In order to complete its activation, this B Cell must receive ‘help’ from T Cells capable of binding the presented antigen in the context of MHC II. Because T Cells have also been selected for ‘Non-Self’ exclusivity, this provides additional insurance that this B Cell was truly activated by a ‘Non-Self’ antigen.  The MHC II :: TCR + CD4 interaction between the antigen-presenting B Cell and the helper T Cell results in activation of the T Cell, that immediately gives activation signals (cytokines) back to the B Cell.


Keep in Mind the Big Picture!

To summarize with an example:

  1. A bacteria gets into the host
  2. B Cells with BCRs capable of binding any part of that bacteria catch ahold of it
  3. These B Cells gobble up the bacteria (endocytosis)
  4. Inside the B Cell, the bacteria is killed and broken into a bunch of little pieces
  5. The little bacteria pieces are picked up by MHC II molecules
  6. MHC II molecules move to the cell surface and ‘present’ antigen
  7. T Cells with TCRs capable of binding this bacteria piece within MHC II, do so
  8. These T Cells become activated, proliferate and produce activation factors (cytokines)
  9. These activation factors trigger the B Cell to go on proliferating and changing into Plasma Cells.

10. Plasma Cells no longer make BCR on the surface, they make a soluble form of that BCR, called Antibody, and spew that forth in great amounts.

11. Antibody can coat, gum up, and signal the disposal of bacteria all over the body.

Resting and Activated T Cells from “Immune System History” by Dr. Harry Louis E. Trinidad 
All that ER expansion is to accommodate the heavy load of secreted protein this cell will churn out.



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Posted by on December 8, 2013 in Uncategorized


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Antigen Presentation #1: Macrophages

Antigen Presentation

Presentation by Macrophages

Macrophages (‘Big Eater Cells) exist in a variety of flavors with slightly different functions throughout the body. For out purposes, we’ll just consider a macrophage that is responding to some inflammation. Like a number of other cells, macrophages can react to inflammatory signals generated by damaged cells at the site of a wound. Once there, the macrophages will eat up (by phagocytosis) whatever they find : cellular debris, dirt, bacteria, anything.

ImageOnce they take up this material, the macrophages will begin to ‘Process and Present’ them. Processing involves the digestion of internalized material by fusion of the phagosome with enzyme-containing lysosomes and reactive-oxygen-species-containing peroxisomes. This treatment breaks up materials into small units that can be bound by Major Histocompatability Complex (MHC) Class II molecules and transported to the cell surface. In this way, the MHC ‘presents’ antigens(Ag) such that they can be tested for binding with the T Cell Receptors (TCRs) on T Cells.

MHC Class II molecules with Ag can be engaged by TCRs with the aid of CD4 acting as a stabilizing molecule between the T Cell and the MHC. However, only TCRs that can soundly bind the MHC+Ag will trigger a reaction that promotes the T Cell to become activated. These activated CD4 Helper T Cells will proliferate and begin secreting molecules (cytokines) that activate other cells in turn. The interaction of T Cells with APCs occurs across a highly organized ‘immune synapse’ where numerous cell-cell recognition molecules come together (see the fluorescent microscopy image below)


      Borrowed from

Helper T Cells provide help to a number of other, effector cells, such as B Cells, Killer CD8 T Cells (also called Cytotoxic T Cells, or CTLs) and macrophages.

It is important to recall that T Cells, like B Cells have undergone negative selection during development so that they only bear TCRs that can bind Non-Self material. Therefore, although self molecules may be presented by APCs, there should be no corresponding  T Cells that recognize these Ags.

Keep in Mind the Big Picture!

To summarize with an example:

  1. A bacteria gets into the host through some wound
  2. The wound stimulates inflammation recruiting Macrophages
  3. Macrophages gobble up the bacteria (endocytosis) in a non-specific manner
  4. Inside the Macrophage, the bacteria is killed and broken into a bunch of little pieces
  5. The little bacteria pieces are picked up by MHC II molecules
  6. MHC II molecules move to the cell surface and ‘present’ antigen
  7. T Cells with TCRs capable of binding this bacteria piece within MHC II, do so
  8. These T Cells become activated, proliferate and produce activation factors (cytokines)
  9. These activated T Cells can now go on to ‘help’  (primarily) B Cells and CD8 ‘Killer’ T Cells  do their jobs

The Immune Synapse


HTLV-I capsid protein (Gag) at the cell-cell 
junction. Tubulin-alpha (green), HTLV-I Gag p19 (red). 
Bar = 5 μm. Source


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Posted by on December 8, 2013 in Uncategorized


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