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A Genetics Riddle

Along with his brothers, a soldier goes off to war leaving behind his wife and two sons. Six years later he returns to his family after losing both his brothers in action. Something is different though. His wife suspects something, but can’t put her finger on it. She just knows that something is different about her husband. Over the next two years, the family grows by twins (a boy and a girl) and then another girl. Then, in an auto accident, the husband dies and his widow decides that she can now investigate a hunch she has had for some time without upsetting her husband.

That month, she takes all of her children in for their annual checkup and vaccines, and also asks the doctor to check her blood type along with all of the children.

The results, mailed to her (see below) later that week, give her a start as she realizes her hunch was correct.

What was her hunch? How did she arrive at her conclusion?

Screen Shot 2016-03-27 at 3.16.30 PM.png

 

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Posted by on March 27, 2016 in Uncategorized

 

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Flow Rate

I received an extra credit essay from one of my students based on a question from the textbook that I had to do a little modeling to understand. The question was one about patients with atherosclerosis that could be explained using Poiseuille’s Law. This Law describes the relationship between the flow rate, pressure, radius and viscosity of a liquid flowing through a vessel.

Basically, it is presented as:

Flow Rate = change in Pressure * pi * radius^4* Length of the vessel * viscosity

.                                                                    8

The question asks, ‘why symptoms of myocardial ischemia do not usually occur until ~75% of a vessel has been occluded.’

The easy answer is that that is the cutoff after which the amount of blood required to provide Oxygen sufficient for the heart’s metabolism is insufficient. However, this can be visualized qualitatively simply by graphing the equation. To do this, I made up a quick spreadsheet and just plugged in ‘1’ for all the variables, then solved for the flow rate. From here, I simply plugged in fractions into the radius variable.

Here’s the raw data:

Screen Shot 2015-05-08 at 5.00.16 PM

1.00 – 0.75 (i.e. a 75% blockage) = 0.25 is the number from the question. Here’s the analysis:

Screen Shot 2015-05-08 at 5.02.20 PM

Note how the Flow Rate has dropped to essentially ZERO when the radius is occluded 75%.

There may be more to this, but I think that just looking at this analysis of the equation answers a lot.

ps – I just spent a hell of a lot of time and effort messing around in the terminal of my mac changing the screen capture file type all to realize that it wasn’t my mac that was the problem at all – I simply was not using the largest image type available in wordpress and then tried to scale up my image after it was inserted – don’t do this. You lose all of your image quality.

 
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Posted by on May 8, 2015 in Uncategorized

 

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ABO+/- : Micro -or- Patho extra credit opportunity

Blood transfusions were first successfully accomplished by Richard Lower in the 1660s. However, like many scientists, he lucked into the right system by using dogs for his experiments. Although there are a number of canine blood types (Dog Erythrocyte Antigens, or DEAs),  only one type, DEA 1.1 leads to severe hemolytic reactions – and only upon secondary transfusions. Therefore, his experiments were very successful, however, they were not easily repeatable in humans for many years.

Lower’s account (as I’ve pilfered from his Wiki page because it was not cited) is as follows:

“…towards the end of February 1665 [I] selected one dog of medium size, opened its jugular vein, and drew off blood, until … its strength was nearly gone. Then, to make up for the great loss of this dog by the blood of a second, I introduced blood from the cervical artery of a fairly large mastiff, which had been fastened alongside the first, until this latter animal showed … it was overfilled … by the inflowing blood.” After he “sewed up the jugular veins,” the animal recovered “with no sign of discomfort or of displeasure.”

The ABO blood typing system has been used since its discovery by Karl Landsteiner in 1901 to allow for life-saving transfusions following accidents, surgery, or to treat other conditions. Classification into the four blood groups most common today, (A, B, AB, and O) was soon afterwards achieved by the efforts of Jan Jansky and his massively significant mustache. The additional understanding and detection of the Rhesus antigen in 1937 with Alexander Wiener, further improved success with blood transfers.

Given the following blood typing card, explain the reactions you are seeing and how this indicates blood type. Also, what is meant by ‘Anti-D’?

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Posted by on May 1, 2015 in Uncategorized

 

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The Hardest Working Organ of the Body

Like the James Brown of the Body

As I started grading the first Pathophysiology Exam on Cardiovascular Function in Health and Disease, the first material I saw was in answer to my questions about the flow of blood through the heart and to the rest of the body. I can’t say that I was happy with the results, but I want to emphasize that this is very basic material that we discussed and outlined in our class, but that I had also assumed was covered in Anatomy and Physiology last semester. After two passes through this material, it should be easily accessible in every student’s mind.

As a reminder of these functions and the flow of blood, here is Khan Academy’s summary of this material:

I believe that that is important foundational material, and you may have a pop quiz on it at any time.

stay on the scene

 
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Posted by on February 21, 2015 in Uncategorized

 

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Virus, Vaccine and Passive Antibody Therapy

The immune system is a many-layered construction that protects the body through barrier defences, additional non-specific responses including phagocytosis and chemokines, an antibody-mediated humoral response capable of neutralizing viral particles, and a cellular response for eliminating infected cells.

Ebola: Disease and Response

mapEbola is a viral disease first identified during a first appeared in 1976 in two simultaneous outbreaks, one in Nzara, Sudan, and the other in Yambuku, Democratic Republic of Congo.  It is reasonable to suspect that Ebola has infected humans prior to this time without being identified specifically. This is a reasonable assertion because, like the first, all subsequent outbreaks have occurred in remote areas of Western African countries that are largely isolated. Although infamous for its lethality, this remoteness has proved self-limiting in terms spread.

The current epidemic has defied these rules resulting in escape from the remote areas of West African villages to larger population centers, and for the first time ever, even resulting in at least one case presenting in the United States. (citation)

In general, although viral infections are not treatable by classical antibiotics, vaccines against these types of organisms have been largely successful. Although it is impossible to know exactly why a specific vaccine works, it is reasonable to assume that a humoral response (i.e. mediated by antibodies) is involved in most cases as antibody titer correlates well with protection.

I the case of Ebola, there is data regarding the type of immune responses mounted by patients who have survived the disease compared to those who have not. Baize et al report that “early and increasing levels of IgG, directed mainly against the nucleoprotein and the 40-kDa viral protein, were followed by clearance of circulating viral antigen and activation of cytotoxic T cells” in survivors of disease. While “fatal infection was characterized by impaired humoral responses, with absent specific IgG and barely detectable IgM.” Again, this supports the idea that an effective humoral response is key to protection.

More evidence of the centrality of the humoral response comes from data published by Villinger, et al (citation) showing that “IL-6 levels are unusually low among fatal cases.” They suggest that this points to a deficiency of the endothelial cells that produce this cytokine leading to failure to protect. An alternative explanation may be that macrophages, which are key targets of ebola infection – and are producers of IL-6, are also failing to respond appropriately due to their involvement as targets. This leads to an obvious defect in immune response as IL-6 supports the growth of B cells and is antagonistic to regulatory responses (i.e. regulatory T cells).

If antibodies are so important to response, what are the targets of these antibodies and what issues are there related to this response?

Ebola Virus:

Eboal5Ebola has only one known surface protein found on virions and infected cells. It is presumed that this protein, a ‘sugar-coated’ glycoprotein (GP), is what enables virions to adhere to target cells, a vital first step in the infection of host cells by animal viruses. As neutralizing immunity against viruses is presumed to be a result of the opsinization of viral particles by antibody, the Ebola GP is the obvious target of these antibodies. However, there are still a number of epitopes (regions of the protein to which immune reactions develop) on the GP protein to which antibodies bind. And, furthermore, two versions of GP are made, one in the viral envelope (membrane) and one that is secreted from infected cells. Together, this means that there are a lot of different spots for antibodies to bind, and some spots may be better for protective immunity, while others have no protective effect at all.

Vaccines against ebola are currently being developed with the hope of bringing these to affected areas to either prevent – or at least control- outbreaks at their source. The benefits of developing an effective vaccine include actively inducing life-long immunity.

A second method of fighting disease is to treat with previously generated antibodies in a way that the virus is neutralized, but life-long protection is not induced. One way of accomplishing this treatment is by harvesting serum from patients who were infected, but survived the disease. This has obvious limitations logistically and there is insufficient data on these treatments to know whether they were actually helpful in treating patients. Another way to transfer this sort of ‘passive’ immunity is by making large amounts of a single antibody in cell culture. These ‘monoclonal’ antibodies are highly standardized and can be produced in very large quantities.

A number of monoclonal antibodies targeting different epitopes on the Ebola GP have been developed and show protective effects when administered after viral exposure (i.e. therapeutically). One example of this kind of therapy is ZMapp  from Mapp biopharmaceutical. In studies with animals, they found that “a combination of monoclonal antibodies (ZMapp), optimized from two previous antibody cocktails, is able to rescue 100% of rhesus macaques when treatment is initiated up to 5 days post-challenge.”

Treatment of Ebola patients with Convalescent Serum

Treatment of Ebola patients with Convalescent Serum

I’ve written before in this space about one of the challenges that antibody treatment against ebola. Because ebola infects macrophages as one of its targets, and because one of the jobs of macrophages is to clear opsonized (antibody-coated) particles, ebola appears to have co-opted this function as a mechanism for penetrating and infecting cells. This characteristic is termed Antibody-Dependent Enhancement (ADE) of infection and has been shown to increase the infectivity of the embryonic kidney cell line, HEK-293, in vitro (Takeda et al 2003). Reportedly, the mechanism for this enhancement is via the complement protein, C1q, and receptors on the host cells.

Together, these data beg the question of whether antibody treatments, such as ZMapp, or vaccines leading to humoral responses will be helpful or harmful in the treatment and protection of patients.

“On 11 August, a group of experts convened by WHO reached consensus that the use of experimental medicines and vaccines under the exceptional circumstances of the Ebola epidemic is ethically acceptable.” So, we may find out the answers to these questions much sooner than we would otherwise expect.

 
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Posted by on November 5, 2014 in Uncategorized

 

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Blood and Genetics

Blood type is a fun and easy way to get to know genetics and learn some practical applications. In the simplest of ways, the three basic blood types are A, B and O.

A good place to brush up on blood type genetics is wikipedia, which has an excellent article on the topic.

Once you’ve reviewed this material (if you need to), then go to nobelprize.org to play a game about blood transfusions (all based on simple blood type genetics).

While you’re on the site, read a little about Alfred Nobel and why it was so important to him to have a lasting positive legacy.

 
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Posted by on November 3, 2014 in Uncategorized

 

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BLyS Sequence Analysis

I’ve been playing with some sequence analysis and phylogentic tree construction programs recently because I would like to introduce these sorts of data analysis into my biology classes. As a sample protein, I decided to use BLyS / BAFF, a protein important in regulating B Cell numbers. I’ve always wondered about the origin of this kind of molecule, since working on it in grad school, and this seemed like a decent way to get some ideas about where it might come from.

The first thing I did was go to the NIH’s National Library of Medicine website: http://www.ncbi.nlm.nih.gov

It’s easy to search for any protein / gene / whole genome you are interested in examining. Knowing that BLyS is vital in humans and mice, I chose to start with the human sequence. I retrieved it as the following:

>gi|20196464|dbj|BAB90856.1| BLyS [Homo sapiens]
MDDSTEREQSRLTSCLKKREEMKLKECVSILPRKESPSVRSSKDGKLLAATLLLALLSCCLTVVSFYQVA
ALQGDLASLRAELQGHHAEKLPAGAGAPKAGLEEAPAVTAGLKIFEPPAPGEGNSSQNSRNKRAV

The easiest tool to find similar proteins in other animals is the Basic Local Alignment Search Tool for proteins, or BLASTp. Just using default settings, I pasted the sequence in the search field and hit go. (note, I actually just used the accession number, not the whole sequence)

Image

This retrieved tons of proteins with similar sequences from the vast database of sequence information, from which I chose several model species. One thing I wanted to do was to include several primates as a sort of internal calibration (assuming that they would all have very similar sequences compared to more distantly related species). I also wanted to get a few animals’ sequences who are quite distantly related to humans (frog and ground tit fir that bill)

Once I had a list, I put them all into a single text file and then used that in a second program. This time, I decided that the best ‘multiple alignment tool’ would be CLUSTALX. It’s been around for a while and can create data in a number of different forms. Besides, it’s free and versions are available for both mac and PC.

Again, for starters, I just accepted the default parameters and did a quick alignment:

Image

Obviously, there’s something odd about the canid familiars (dog) sequence, but before I did anything about that, I just wanted to see what a phylogenetic tree looked like. This is another thing that Clustal does well, it will export your sequence alignment as tree data in a number of formats, then I could plug that data into one final program. This last is a web based program that I access through a french site (but you can probably find it in a number of places). The program is called DRAWGRAM. It accepts alignment data and outputs a graphical tree representation of the alignment.

This is an important logical step… What I’m doing is asking for a family tree of sorts to be displayed that represents the relationship of the sequences I provided. We might want to assume that this also tells us how related the organisms that have these proteins are – and that’s not wrong, but it’s also not thorough as we’re only using ONE protein to make that assumption.

Here’s my first tree:

Image

Note how isolated Canis is on this representation.

Finally, I went back and truncated the Canis sequence to a place where I suspect the protein actually starts – my sequence from the NCBI gave me a string of Amino Acids at the front of the protein that I think are probably not there, but just got added by some computer algorithm without proper human oversight.

Once I did that Canis (by the way, I remained the sequence ‘DOG’ so I was sure it was the new one) fell in line with a sequence more similar to that seen in cats (felis):

ImageThat’s it for now. Although I expect that I will dig a little deeper with more animals to see if I can come closer to an ‘original BLyS’.

 References:

  1. Dereeper A., Audic S., Claverie J.M., Blanc G. BLAST-EXPLORER helps you building datasets for phylogenetic analysis. BMC Evol Biol. 2010 Jan 12;10:8. (PubMed)
  2. Dereeper A.*, Guignon V.*, Blanc G., Audic S., Buffet S., Chevenet F., Dufayard J.F., Guindon S., Lefort V., Lescot M., Claverie J.M., Gascuel O. Phylogeny.fr: robust phylogenetic analysis for the non-specialist. Nucleic Acids Res. 2008 Jul 1;36(Web Server issue):W465-9. Epub 2008 Apr 19. (PubMed) *: joint first authors
  3. Felsenstein J. PHYLIP – Phylogeny Inference Package (Version 3.2). 1989, Cladistics 5: 164-166
  4. Larkin,M.A., Blackshields, G., Brown, N.P., Chenna, R., McGettigan, P.A., McWilliam, H., Valentin, F., Wallace, I.M., Wilm, A., Lopez, R., Thompson, J.D., Gibson, T.J., Higgins, D.G. (2007) Clustal W and Clustal X version 2.0. Bioinformatics, 23:2947-2948.
  5. Thompson,J.D., Gibson,T.J., Plewniak,F., Jeanmougin,F. and Higgins,D.G. (1997) The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research, 25:4876-4882.
 
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Posted by on March 7, 2014 in Uncategorized

 

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