Cytokine production by different blood cell types

Spot size and morphology frequently allow researchers to distinguish cytokine production by different cell types within mixed cell populations. For example, when IL-10 production by human peripheral blood mononuclear cells (PBMC) is measured in ELISPOT assays, most of the “antigen-induced” spots are not T cell derived (as would be expected), but are produced by macrophages in response to LPS contamination of the antigen. Such macrophage-derived IL-10 spots are considerably smaller than the IL-10 spots generated by antigen-specific T cells. While the LPS-induced macrophage-derived spots provide no information on specific immunity, the antigen-induced T-cell-derived IL-10 spots do, since they indicate the presence of T regulatory cells. In order to measure the latter, the former need to be excluded from the counting results by setting appropriate size thresholds. ELISPOT image analysis must therefore be capable of distinguishing different spot sizes and morphologies to provide information relevant for T cell diagnostics.

The antigen dose affects the cytokine secretion rate of T cells. Stimulation of a T cell clone with a high dose of the nominal antigenic peptide induces stronger cytokine production in the individual T-lymphocytes (that is, it triggers larger and/or denser spots) than does the stimulation of the same clone with low dose peptide. Therefore, when stimulated with a single antigen dose, as is frequently the case in ELIPSOT assays, high-avidity T-lymphocytes within the PBMC produce larger spots than low-avidity clones. Confirming this notion, increased T cell costimulation was shown to result in increased per cell productivity. In diseases such as HIV, the per cell cytokine productivity can be reduced, resulting in smaller spots. One advantage of ELISPOT assays is their ability to determine whether decreased net cytokine production in disease states is caused by a decreased number of cytokine-secreting T cells or from reduced per cell productivity by unchanged frequencies of T cells. In order to compensate for physiological and pathological variations in per cell productivity, ELISPOT image analysis tools must therefore be versatile, with the ability to permit fine-tuning of the image processing parameters.

More about the limitations of science

Continuing the previous discussion about the limitations of science...

I mentioned that some people insist that the scientific method is the only way through which truth can be discerned. I also mentioned that when they say that, I ask "so what scientific methodology did you use to determine that to be true?"

Unfortunately, that doesn't stop some people from insisting on that claim. Instead of re-examining that belief, some of them say "But it's true! It's obvious!" or words to that effect. The thing is, unless one can demonstrate it to be true [em]using the scientific method[/em], that claim remains unsubstantiated.

Indeed, how would one even begin to test this claim? I ask people that sometimes. Some of the more hard-headed ones say, "Well, you design an experiment to test this belief, and then you conduct the experiment. That's how science works!" However, this answer completely glosses over the question of HOW someone would design such an experiment -- or even if such an experiment is possible.

Some go a bit further, and say "Well, you take all the possible methods of learning knowledge, and then you test every single one of them. That will prove that science is the only means to knowledge." This answer is a bit more complete, but still unsatisfactory. For one thing, it ignores the question of HOW one would identify all the possible paths to knowledge. And second, it dodges the question of how one would test all these methods.

Indeed, how would one know that one has identified all paths to knowledge? For that matter, how would one know that ANYTHING is a means of discerning truth? If the claim is true, then one must first conduct a scientific experiment to establish these things to be true.

What's more, if someone is to claim that science is the ONLY way through which knowledge can be truly obtained, then one must test ALL possible methods under ALL possible circumstances. For if there is even a single situation in which one of these methods can work, then the scientific method is not the sole means to truth.

This is why I'm severely disappointed when people claim that only science holds the keys to truth. It's a statement that sounds neat, but which people often don't examine closely. It has no basis in reality.

The limitations of science

Every now and then, I hear people claim, "The scientific method is the only way that truth can be known!" The problem is that this is a self-refuting claim. It also betrays great naivete about the nature of science.

Self-refuting, you say? Yes indeed. Consider this: If the scientific method is the only way by which truth can be known, then what scientific methodology would one employ to arrive at that truth? The people who make this claim clearly have not performed any such experiment, and so their claim remains unproven. What's more, it CANNOT be demonstrated to be true, since one would have to identify all possible alternatives to science and then demonstrate that none of them are ever valid.

"But it's obvious!" some exclaim. Well, if you claim that it's true by virtue of being obvious, then you're not using the scientific method. Rather, you are claiming that something is self-evident. Ergo, you have implicitly demonstrated that science is not the only means by which truth can be discerned.

I know one guy who says, "The methodology that I use is observation. It's science!" Um, no. Observation is merely the first step in applying the scientific method. It is not an example of scientific methodology. Without the subsequent phases of hypothesis-forming and experimentation, one does not have a scientific method.

Indeed, there are things that science implicitly assumes, and thus, cannot prove. It assumes that the laws of logic and mathematics are valid, for example. As such, attempting to prove these tenets using science would amount to circular reasoning. Now, I'm not about to deny that the laws of math and logic are valid; however, my point is that one cannot use science to prove their validity. That would be begging the question, pure and simple.

Ohm's Law

Ohm's law states that the voltage drop between the ends of a conductor (or resistor) is directly proportional to the current flowing through it, provided the temperature doesn't change. This is commonly stated as V=IR, where V is the voltage drop, I is the current and R is the proportionality constant (or "resistance").

Ohm's Law is not truly a law. There is a popular misconception among many less knowledgeable engineers that Ohm's Law always holds true; however, most substances do not display the proportionality described. In such cases, the voltage and current have a more complex relationship, which is described as a transconductance curve.

Indeed, no real-world device obeys this "law" perfectly. However, Ohm's Law is an adequate approximation for the behavior of many objects, such as strips of metal.

Granzymes

Granzymes are exogenous serine proteases that are released by the cytoplasmic granules within cytotoxic T cells and natural killer cells. Cytotoxic T cells and natural killer cells release a pore-forming protein (perforin) which attacks the target cells. Granzymes can enter through these pores, inducing apoptosis.

Agile manufacturing

The term "agile manufacturing" can have different definitions, depending on how the term is used. As a result, it can be confused with similar and related terms such as "flexible manufacturing" and "just-in-time (JIT) manufacturing." The most useful definition, however, describes it as the ability to achieve rapid changeover between the manufacture of different assemblies. This is extremely helpful in modern contexts, wherein product changeover is frequent.

Agile manufacturing is more of engineering than science; however, we shall mention it here because its effective use ideally requires the application of diverse intelligent disciplines -- computer vision and object recognition, intelligent robotic manipulation, intelligent object sensing and so forth. As such, it can straddle the boundary between science and engineering.

This philosophy also requires tight mechatronic integration, interlinking mechanical, electronic and software design in a synergistic manner.

Histocompatibility

Histocompatibilty is the condition in which the body allows the grafting of tissue or the transfusion of blood without rejection. Ultimately, this means that there is anabsence of interference from the body's immune system.

Histocompatibility is governed by Major Histocompatibility Complex (MHC) molecules. Crudely stated, their purpose is to distinguish "self" from "non-self". More specifically, their primary immunological function is to bind and present antigenic peptides on the surfaces of cells for recognition by antigen-specific T cell receptors.

Controlling and inducing histocompatibility is central to ensuring the success of organ transplants. This field is known as transplantation immunology.

Buoyancy question

Here's a question for people to ponder.

Say you have a boat floating in a swimming pool. Suppose that there's a rock sitting in the boat. Now suppose that someone takes the rock and drops it into the pool.

Does the water level rise or fall? Think about it.

Spot counting and "Zytokinen"

Durch automatische Auszählung mittels Software kann so bestimmt werden, welche spezielle Aktivität die Immunzellen besitzen, was für die Forschung und Diagnostik von Autoimmunerkrankungen, Transplantationsrisiken, Allergien und Infektionskrankheiten nützlich ist, ebenso wie für die Impfstoffforschung.

Cytokines

Cytokines are regulatory proteins that are released by cells of the immune system. These act as intercellular mediators in the generation of an immune response. They can have stimulating or suppressing effects on lymphocyte cells and immune responses.

Well-known examples include the interleukins, interferons and tumor necrosis factors. Cytokine production can be detected using certain laboratory techniques that have been discussed previously.

ELISA illustration

Today, I just wanted to post this link to an animated illustration of the ELISA process. They say that a picture paints a thousand words, and in this case, an animated (and interactive) picture says a whole lot more.

Scientific illiteracy

Scientific illiteracy is a tremendous problem in this country. I'd like to quote the following statements by George Dvorsky (as reproduced in this web essay).

Most of those who live in the West, particularly North Americans, are guilty of an anti-intellectual bias. Scientists are supposed to be nerds, right? And who wants to be a nerd? This sentiment, combined with a general suspicion of science and the predominance of aggressive theological and pseudoscientific memes, has resulted in much of the scientific illiteracy that now pervades our society.

It doesn't help that the educational system is in shambles and without focus, and that fatuous postmodernism and its insistence that nothing can truly be known now dominates many disciplines at most universities. Consequently, too many people wear their ignorance like a badge of honor, as if being clueless about science is something to be proud of.

Memory cells

Memory cells allow the body to remember previous infections and quickly mobilize defenses against them. This is the mechanism by which the human body remembers that someone contracted chicken pox, for example, and which prevents future infections of that type from occuring.

Time is short, so instead of discussing this at greater detail, I'll post a (slightly paraphrased, for brevity) excerpt from a technical paper on this subject.

Single-Cytokine-Producing CD4 Memory Cells Predominate in Type 1 and Type 2 Immunity

The patterns of Ag-induced cytokine coexpression in normal, in vivo-primed CD4 memory T cells has remained controversial because the low frequency at which these cells occur has effectively prevented direct ex vivo measurements. We have overcome this limitation by using two-color cytokine ELISPOT assays and computer-assisted image analysis. We found CD4 memory cells that simultaneously expressed IL-2, IL-3, IL-4, IL-5, and IFN--y to be rare (0-10%). This cytokine segregation was seen in adjuvant-induced type 1, type 2, and mixed immunity to OVA, in Leishmania infection regardless of the Ag dose used or how long after immunization the assay was performed. The data suggest that type 1 and type 2 immunity in vivo is not mediated by classic Thl or Th2 cells but by single-cytokine-producing memory cells.


Alexey Y. Karulin, Maike D. Hesse, Magdalena Tary-Lehmann, and Paul V. Lehmann, The Journal of Immunology, 2000, 164: 1862-1872.

Lymphocytes

Lymphocytes are a category of white blood cell, or leukocyte. The main categories of lymphocytes are B cells and T cells. B cells are responsible for manufacturing antibodies, whereas T cells have more complex roles. Depending on the category of the T cell, it may be responsible for destroying invading cells, for secreting cytokines in response to an antigen, or for activating macrophages and NK-cells. These processes shall be discussed at a later date.

Flow cytometry was previously mentioned, and one application of this technique is in counting specific lymphocyte population. The ELISPOT method was mentioned as well; this technique is used for counting the number of B cells that secrete a specific antibody, or the number of T cells that produce a given cytokine or granzyme.

Cytokines

Cytokines are regulatory proteins that are released by cells of the immune system. They are the means by which cells communicate in the generation of an immune response. Well-known cytokines include prostaglandin, the interleukins, the interferons and the lymphokines.

Cytokines trigger responses by binding to cell-specific receptors located in the cell membrane. This allows a distinct signal cascade to start in the cell, eventually generating biochemical and phenotypical changes in the target cell.

Production of a specific cytokine can be detected using the aforementioned ELISPOT assays, which can also determine the percentage of cells that produce the cytokine in question.

Robotic grasp theory

Robotic grasp theory is a magnificent marriage of science and engineering. The human hand is a magnificent piece of design -- flexible and adaptable beyond belief. It's no surprise that many robotic efforts have attempted to duplicate its capabilities.

Sadly, these efforts often go too far in either extreme. Some attempt to duplicate the human hand too closely. The result? Anthropomorphic robot hands that require hopelessly complex control algorithms, and which lack the fine sensing capabilities of a human hand. These devices are cumbersome, and often impractical to use. Others use primitive parallel jaw grippers, which are decidedly more restricted in their capabilities. Since most parts require some custom tooling in order to be held securely, this greatly limits the range of workpieces that a parallel jaw gripper can hold.

(Other variants exist, such as three-fingered end-effectors. The end results is similar, though -- limited grasp capabilities.)

Velasco et al. offered a compromise in which rapid prototyping technology was used to develop gripper tooling based on CAD models of the parts to be grasped. The CAD capabilities allowed the tooling to be developed rapidly, and with due consideration to part clearances and manufacturing variability. This gave parallel jaw grippers a great deal more versatility than could be achieved using rudimentary tooling, without requiring the complex mechanisms or ocntrol algorithms required of anthropomorphic robot hands.

Vaccine development

The first vaccine was developed by Edward Jenner in an abhorrently crude fashion. Jenner noticed that milkmaids were not readily susceptible to smallpox infections, which had been ravaging the populace. He theorized that they had somehow gained immuniity through regularly exposure to cowpox lesions. In a bold move, Jenner inoculated an eight-year-old boy with scrapings from cowpox lesions, thus proving his theory.

Such an experiment would never be allowed nowadays, due to medical ethics. To Jenner's credit though, it worked, and this paved the way toward stamping out smallpox across the world.

Nowadays, more sophisticated techniques are available for vaccine development. In an earlier write-up, we discussed the ELISPOT technique in brief. Suffice to say that this methodology allows the monitoring of immunological responses to antigen invasion at the cellular level. This eliminates the need for crude experiments with human subjects under coarse conditions.

Asthma and immunology

I suffered a mild asthma attack while cycling to work today, thanks to the influx of cold air into my lungs. So, I decided to write a little about asthma.

Asthma is an immunological disorder in which the patient suffers respiratory difficulty. In this disease, the bronchioles in the lungs are narrowed by inflammation, thus requiring treatment with bronchodilators. The symptoms include wheezing and shortness of breath, which can prevent the afflicted person from engaging in prolonged exercise.

There are several theories which purport to explain the rising incidence of asthma. One places the blame on air pollution. Another purports that the hygenic conditions in the First World cause reduced bacterial exposure, causing inadequate stimulation of the immune system during early childhood. Yet another suggests that the problem is due to non-exposure to parasites such as tapeworms, since the cells which combat these parasites are the same ones which trigger the asthmatic reaction.

T cell clone avidity

Tonight I decided to cite the abstract of a published paper that I got my hands on. It's about memory cells, which are used to retain a record of previously encountered antigens. These cells allow the immune system to respond more rapidly to subsequent invasions.

The patterns of Ag-induced cytokine coexpression in normal, in vivo-primed CD4 memory T cells has remained controversial because the low frequency at which these cells occur has effectively prevented direct ex vivo measurements. We have overcome this limitation by using two-color cytokine enzyme-linked immunospot assays and computer-assisted image analysis. We found CD4 memory cells that simultaneously expressed IL-2, IL-3, IL-4, IL-5, and IFN--y to be rare (0-10%). This cytokine segregation was seen in adjuvant-induced type 1, type 2, and mixed immunity to OVA, in Leishmania infection regardless of the Ag dose used or how long after immunization the assay was performed. The data suggest that type 1 and type 2 immunity in vivo is not mediated by classic Thl or Th2 cells but by single-cytokine-producing memory cells.

(Maike D. Hesse, Alexey Y. Karulin, Bernhard O. Boehm, Paul V. Lehmann, and Magdalena Tary-Lehmann. The Journal of Immunology, 2001, 167: 1353-1361.)

Biotech

Biotechnology is the marriage of engineering and technology principles to the biological sciences. Common applications include the development of new microorganisms or biological substances for industrial applications. These can include hormone production, waste cleanup, bioconversion of organic wastes, genetic engineering of new organisms, drug manufacturing and so forth.

Biotechnology does not necessarily use living organisms. For example, it can focus on DNA chips in genetic research, or radioactive medical tracers. It can even use active cells from living organisms -- but not the organism itself -- such as when human lymphocytes are used in vaccine development.

Among the most exciting of biotech fields is the engineering of new microorganisms, with the goal of producing insulin or antibiotics cheaply and efficiently. Other applications include the development of transgenic plants and animals, in which foreign genes are introduced externally, so as to produce enhanced organisms (e.g. cows that produce extraordinary quantities of milk).