What are T-cells? It is our big curiosity what T-cells are. Have you ever heard of them?
T-cells are a type of white blood cell that circulate around our bodies (yes they are in your blood stream this very right minute!), scanning for cellular abnormalities and infections that might affect your ability to heal.
What is the role of T-cells in the immune system? Why are they named that way?
T-cells are essential for the human immune system and response. The reason why they are called T-cells is because they mature in the thymus (even some others also mature in the tonsils). T helper cells (TH cells) assist other white blood cells in immunologic processes, including maturation of B-cells into plasma cells and memory B-cells, and activation of cytotoxic T-cells and macrophages. Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or assist in the active immune response. These cells can differentiate into one of several subtypes, including TH1, TH2, TH3, TH17, Th9, or TFH, which secrete and produce different substances called cytokines to facilitate different types of immune responses. Signaling from the APC directs T-cells into particular sub-types. Helper T-cells, or Th cells, coordinate immune responses by communicating with other cells. Some stimulate nearby B cells to produce antibodies, others call in microbe-gobbling cells called phagocytes, and still others activate other T-cells.
T-cells are the key to the immune system and they represent the core of adaptive immunity, the system that tailors the body’s immune response to specific pathogens. The T-cells are like soldiers who search out and destroy the targeted invaders.
According to the National Institute of Allergy and Infectious diseases, T-cells aid the normal processes of the immune system. If T-cells fail to function properly, asthma, certain autoimmune diseases—including Type 1 diabetes—or the growth of cancers may result. NK T-cells get their name because they are a kind of T lymphocyte that carries some of the surface proteins, called “markers,” typical of NK T- cells. But these T-cells differ from other kinds of T-cells. They do not recognize pieces of antigen bound to self-MHC molecules. Instead, they recognize fatty substances (lipids and glycolipids) that are bound to a different class of molecules called CD1d. Scientists are trying to discover methods to control the timing and release of chemical factors by NK T-cells, with the hope they can modify immune responses in ways that benefit patients.
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“T Cell.” MedicineNet. N.p., n.d. Web. 16 Feb. 2015.
“National Institute of Allergy and Infectious Diseases (NIAID).” National Institute of Allergy and Infectious Diseases (NIAID). N.p., n.d. Web. 17 Feb. 2015.
An Innovative Approach to Immunotherapy [Developing the Immune System]
Therapeutic agents for markets with high under media needs: infectious diseases.
Initial discovery: Synthetic peptides.
The need: Viral infections and cancers dysregulate the immune system, which allows progression of the disease. A new approach to therapy is needed to modulate the immune system.
Approach: Design peptide drugs that bind lectin-type receptor to activate cellular processes at very low concentrations.
Triggering cellular processes and then metabolized. This means high avidity and low toxicity.
Focus: Cells must be activated to destroy aberrant cells. Modulation of immune system is achieved by identification of peptides that bind to specific areas of a cell.
Phage sequence: PIII Protein. DNA synthesis: Amino acids that act as sugars in the system. Peptide sequences that mimic sugars were also identified by computer modeling of interaction with lectin as receptor analogs.
In order to complete the reaction binding energy is needed= –ΔG.
(Negative values for energy input).
Lectin type receptors are important in the process of detecting specific sialic acid-containing.
Function: Sialic acid-rich glycoproteins (sialoglycoproteins) bind selectin in humans and other organisms. Metastatic cancer cells often express a high density of sialic acid-rich glycoproteins. This over expression of sialic acid on surfaces creates a negative charge on cell membranes. This creates repulsion between cells (cell opposition) and helps these late-stage cancer cells enter the blood stream.
Solid-phase binding assay for peptide-lectin interaction.
Blue color predicts the amount of peptide you created. The peptide binds as a monomer, and the phosphorylation.
Results: Intracellular fluorescence color in tests predicts the formation.
Benefits: Reduce HIV-related morbidity and prolong survival. Improve quality of life. Maximally and durably suppress viral load. Prevent HIV transmission. HIV inhibition, and inhibits replication.
Restore and Preserve Immunological System.
Test: In-vitro assays proved that svH1C eliminated HIV culture of peripheral blood mononuclear cells present in the blood-antibodies.
Glioblastoma: Survival of mice when tumor was exposed to brief radiation. The more phosphorylated, the more active the receptor to activate the cell. Dephosphorylated removes its activity to serve as a receptor. Inhibitory receptors or decrease this allows the activated signals to be expressed even more.
Glioblastoma multiforme (GBM) is the most common and most aggressive malignant primary brain tumor in humans, involving glial cells and accounting for 52% of all functional tissue brain tumor cases and 20% of all intracranial tumors. Despite being the most prevalent form of primary brain cancer
Glioma cells: Cells with same histological features.
A glioma is a type of tumor that starts in the brain or spine. It is called a glioma because it arises from glial cells. The most common site of gliomas is the brain. Gliomas make up ~30% of all brain and central nervous system tumors and 80% of all malignant brain tumors.
Brain microglial cells, and dendritic cells. Promotes maturation of dendritic cells in the brain.
Susavion compounds should be effective in immunotherapeutic treatment of diseases in which the immune system is suppressed helping the body to defend itself.
Further research: Delivery system: epithelium absorption and how to get absorption into the digestive system.
Thin Layer Chromatography [Analysis of Analgesic Drugs]Abstract: In this experiment, a thin-layer chromatography (TLC) was used to determine the composition of various over-the-counter analgesics. (Acetaminophen, Acetylsalicylic Acid, Caffeine, and Acetylsalicylic Acid). Chromatography is a technique that we used to separate and identify individual components in a mixture. These chromatography techniques focused on the fact that components of a mixture tend to move at different speeds along the flat surface from the paper used to differentiate them from each other. The different rates of movement were the results of differing attractions of the components to the coating material compared to the tendency of the components to remain in the moving fluid. The results obtained were compared using a Co-Spot method to ensure their identity.
IntroductionThin-layer chromatography is one of the easiest of the many chromatography techniques. A thin layer of a suitable solid substance is coated on a sheet of glass or plastic. A very small sample of the mixture to be analyzed is “spotted” onto the sheet. By immersing one edge of the sheet in an appropriate liquid developing solvent, the solvent is drawn up the sheet by capillary action, and the compounds of interest are carried along at different rates, effectively separating the components. This is commonly called “developing” the plate. After the plate has been developed, it is examined under ultraviolet (UV) light, which allows you to note the location of the spots. Experimental conditions in TLC include the solvent system used to dissolve the compounds, the absorbent coated onto the TLC plate, the thickness of the absorbent layer, and the relative amount of the material spotted onto the plate. Under an established set of experimental conditions, a given compound always travels a fixed distance relative to the distance the solvent front travels. This ratio of the distance the compound travels to the distance the solvent front travels is called the Rf value. The symbol Rf stands for “retardation factor,” or “ratio-to-front,” and it is expressed as a decimal fraction. The equation is shown below: AnalgesicsWe obtain half a tablet of four different analgesics. The group took each analgesic and and crush it to dissolve. Transferred each crushed half-tablet to a DRY, labeled 50-mL beaker. Using a graduated cylinder, add 5 mL of the Solution Solvent, which was an ethyl acetate-ethanol-acetic acid mixture, to each of the crushed half tablets, and swirl to mix. Then heat each of them gently for a few minutes on a hot plate set at low. Not the entire tablet was dissolved because the analgesics usually contain an insoluble binder. Some of them also contained inorganic buffering agents or coatings that are insoluble in this solvent mixture. After heating the samples, we swirl them again to mix and then allowed the insoluble particles to settle.Spotting the TLCPlatesThe group obtained from the hoodfour capillarymicropipettes to spot the plates with the four solutions of analgesic prepared above. The instructor showed how the plates were supposed to be set. This procedure was repeated for every compound (Total of six spots).. The standard reference mixture contains acetaminophen, aspirin, caffeine and acetylsalicylic acid.Developing the TLC PlatesAfter spotting your plates, the group obtained a jar for use as a development chamber. Using a graduated cylinder, we measure about 10-15 mL of Development Solvent (0.5% glacial acetic acid in ethyl acetate) and poured it into the jar. Making sure it didn’t go above the reference line or the samples would have been dissolved off the plate into the solution instead of developing. When the solvent front had risen to a level about 0.5 cm off from the top of the plates, we removed the plate from the jar and, using a pencil, mark the position of each solvent front. Set the plate on a piece of paper towel in your bench-top hood to dry. We saved the developing solvent into the jar for our ‘Co-Spot Method’.Observing the TLC Sample & Reference Plates: UV visualization.
We then, observe the reference plate under the UV lamp. We also, sketched the spots on the plate using the ruler; we measured, in centimeters, the distance traveled by each spot on both sample plates as well as the distance traveled by the solvent front. (to get our Rf values). See Lab Notebook. Also, these values were used to compare the identity of the unknown compound.
This was performed after the UV visualization was performed. We placed the second plate called the “Co-Spot Method” in the jar located on the hood (pre-heated). The yellow spots appeared, which were used to ensure the result already established. Below a picture shows these results.
Results and Conclusions
Below, pictures from the actual results obtained performing this experiment.
P.1 This reference plate shows the results obtained after looking at the plate under UV light. The spots localized were sketched and our first matching spot to the unknown #14 compound showed similarities to acetylsalicylic acid underlined in this picture. The Ref line showed that every compound was included in the reference mixture.
P.2 This reference plate shows the yellow/brownish spots that appeared after the plates were placed into the jar containing the iodine crystals and after the UV visualization was performed. The one on the left is the ‘Co-spot Method’, which contained:
Unknown: Acetylsalicylic Acid
The one on the right side is the first reference plate.
Contained: the 6 mentioned Ref Compounds.
Results: According to data obtained the unknown compound #14 was Acetylsalicylic acid.
Explanation: If the unknown compound would have been ibuprofen then the first unknown would have matched up, but it didn’t. Ibuprofen and acetylsalicylic acid were used in the first plate to compare similar results.
With this lab, a thin-layer chromatography (TLC) was used to determine the composition of various over-the-counter analgesics, and how they will be identified using chromatography techniques (Acetaminophen, Acetylsalicylic Acid, Caffeine, and Acetylsalicylic Acid). These chromatography techniques focused on of the fact that components of a mixture tend to move at different speeds along the flat surface from the paper used to differentiate them from each other. We identified one the compounds as being Acetylsalicylic Acid.
1. What happens if the spots are made too large when preparing a TLC plate for development?
When the results have to be read, the spots will be on top of each other (Overlapping) and it will make very hard to read the reference plate.
2. What happens if the spots are made too small when preparing a TLC plate for development?
It is preferable that they are made small-sized as long there is enough for development and they are consistent to each other. Otherwise, if they are tiny-to-almost invisible then the results will be harder to read.
3. Why must the spots be above the level of the development solvent in the developing chamber? Because the absorption of the solvent has to be equally distributed along the spots, and they have to wait for the solvent to go up (mobile phase). If they were lowered down, they will be dissolved into the solvent and the development wouldn’t work.
4. What would happen if the spotting line and positions were marked on the plate with a ballpoint pen?
That will be a huge mistake because the ink from the pen will stick to some of the solvent and will be traveling itself through the paper. Making the results impossible to read.
The procedure will have to be redone if this happened.
5. Is it possible to distinguish two spots that have the same Rf value but represent different compounds? Give two different methods.
The first method will be to make a different plate with three reference spots.
For our experiment, we tried this extra reference plate. Called “Co-Spot Method’ and it worked just fine.
Repeating procedures for making the reference plate, three reference spots are marked down on the line: Unknown, Unknown plus Known, and finally the Known one.
Unknown: You spot the unknown substance provided by instructor.
Unknown +Known: Here you spot again the one provided by instructor (#14) and one of the known to compare if the result will match.
Known: You spot the only one you are sure about because there will find out if the assumptions are correct.
Another one would be: Gas chromatography. In gas chromatography, the mobile phase (or “moving phase”) is a carrier gas, usually an inert gas such as helium or an unreactive gas such as nitrogen. The stationary phase is a microscopic layer of liquid or polymer on an inert solid support, inside a piece of glass or metal tubing called a column (an homage to the fractionating column used in distillation). The instrument used to perform gas chromatography is called a gas chromatograph (or “aerograph”, “gas separator”). This would be very similar to the paper chromatography but instead of the solvent for the moving phase you will use the gas to get the results.
Techniques 20: 1 to 5
1. A student spots an unknown sample on a TLC plate and develops it in dichloromethane solvent. Only one spot, for which the Rf value is .95, is observed. Does this indicate that the unknown material is pure compound?
No, because you will have to make mark a different spot with the reference to make a fair comparison. Only if more than one spot separated from the original spot you are trying to compare, then the compound would be impure. If the student only spots one unknown sample then it is expected it there will be only one Rf value.
What can be done to verify the purity on the sample using thin-layer chromatography? When dealing with compounds of similar properties, such as isomers, multiple developments of the TLC plate with a fairly nonpolar solvent may be necessary to effectively separate compounds and so to determine purity.
2. You and another student were each given an unknown compound. Both samples contained colorless material. You each used the same brand of commercially prepared TLC plate and developed the plates using the same solvent. Each of you obtained a single spot Rf=.075. were the two samples necessarily the same substances? How could you prove unambiguously that they were identical using TLC?
Not necessarily the same. We could try making another plate with those different compounds on the reference line, and comparing them with the compound that we think it is. That will give us two unknowns and one known then, we can compare the Rf values to see if they actually match.
3. Each of the solvents given should effectively separate one of the following mixtures by TLC. Match the appropriate solvent with the mixture that you would expect to separate well with that solvent. Select your solvent from the following: hexane, methylene chloride, or acetone. You may need to look up the structures of the solvents and compounds in the handbook. Polar will dissolve a polar.
- Use methylene chloride for the TLC with 2 – phenylethanol and acetophenone.
- Use Hexane for the bromobenzene and p-xylene.
- Acetone for benzoic acid, 2,4-dinitrobenzoic acid, and 2,4,6-trinitrobenzoic acid.
4. Consider a sample that is a mixture composed of biphenyl, benzoic acid, and benzyl alcohol. The sample is spotted on a TLC plate and developed in a dichloromethane-cyclohexane solvent mixture. Predict the relative Rf values for the three components in the sample. (hints. See table 19.3) Biphenyl: nonpolar. Benzoic acid: Neutral/slightly nonpolar. Benzyl alcohol: Polar. Therefore, the polar compounds will interact more with the solid phase and will not travel as far (lower Rf). Rf will increase with decreasing polarity and the order will likely be polar compounds with the lowest Rf and nonpolar compounds with the highest Rf. Benzoic acid will have the lowest Rf (polar carboxyl group), followed by benzyl alcohol (slightly polar), and biphenyl (nonpolar) with the highest Rf.
5. Consider the following errors that could be made when running TLC. Indicate what should be done to correct the error.
a. A two-component mixture containing 1-octane and 1,4-dymethylbenzene gave only one spot with an Rf value of .95. The solvent used was acetone.
I will try testing the results with other methods again because even with the same Rf values, they show up differently under the detection methods. Here, the solvents have different polarities, affecting the way the compound attaches to the solvent. We can also try using a less polar solvent.
b. A two-component mixture containing a dicarboxylic acid and tricarboxylic acid gave only one spot with an Rf value of .05. The solvent used was hexane.
Hexane is a nonpolar molecule and the other two are polar molecules. Therefore, it is unlikely they will react with the solvent. Use a more polar solvent will be helpful.
c. When a TLC plate was developed the solvent front ran off the top of the plate.
The distance traveled by the solvent will be inaccurate, and then we’ll have to set a new plate since all the Rf values will not be valid.
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