What is the role of the ciliated epithelia as a defense barrier of the mammalian respiratory system?

• It acts to move mucus that has trapped bacteria out of the upper respiratory tract via the mucocociliary escalator.

List four examples of “exterior” innate immune defense mechanisms.

• lysozyme, external skin, epithelial cells joined by tight junctions, mucociliary escalator, fatty acids, low pH, defensins, cryptidins, normal flora microorganisms.

Name 3 different types of “pathogen associated molecular patterns” of bacteria that result in the activation of the “acute phase response” of the innate immune system.

• Lipopolysaccharide, lipoteichoic acid, peptidoglycan, lipoproteins, flagella, unmethylated CpG DNA sequences.

Adaptive immunity that involves CD4+ lymphocytes and a Th2-like response results in what type of immune effector molecules or cells? What type of bacterial pathogenic strategy are these effector molecules most effective against.

• A humoral or antibody response and this is most effective against extracellular pathogens.

How does mucus production function as a defense mechanism in the upper respiratory tract of mammals, including humans?

• Mucus is produced by cells lining the upper respiratory tract, the mucus entraps bacteria, the mucus entrapped bacteria are moved out of the respiratory tract by ciliated epithelial cells (upward motion of the cilia) into the mouth where they are swallowed and deposited into the stomach. The lungs.

How do members of the normal bacterial flora serve as a defense mechanism against potential bacterial pathogens?

Name 2 ways in which the activated complement system defensively works during an innate immune response?

Name 2 types of professional phagocytes and describe in general terms how they engulf and destroy microorganisms?

Name 3 different types of bacterial PAMPs that stimulate the acute phase response of innate immunity?

Adaptive immunity that involves CD4+ lymphocytes and a Th1-like response results in what type of immune effector molecules or cells? What type of bacterial pathogenic strategy are these effector molecules or cells most effective against?

Name 2 ways in which specific antibody functions defensively against a bacterial pathogen?

List three examples of “chemical” exterior innate immune defense mechanisms.

• fatty acids, enzymes (lysozyme or pepsin), low pH, antibacterial peptides (defensins or cryptidins)

Name 2 different types of bacterial “pathogen associated molecular patterns” that result in the activation of the “acute phase response” of the innate immunity.

• LPS, Lipoteichoic acids, lipoproteins or lipopeptides, peptidoglycan, flagella, unmethylated GC sequences

What are the surface receptors on macrophages, PMNs, and epithelial cells that PAMPS bind?

• Toll-like receptors or TLRs

What general types of chemical messengers are released from these cells that modulate and mediate the “acute phase response”?

• Pro-inflammatory cytokines or cytokines

Adaptive immunity that involves T-cell receptor recognition on CD4+ lymphocytes and a Th2-like response results in what type of immune effector molecules or cells? What type of bacterial pathogenic strategy are these effector molecules or cells most effective against.

• Antibody (Humoral immunity). Extracellular pathogens

Experiment 8A

1. The living cells adhered to the bottom of the plate and looked dark and small. I saw what looked like organelles as I squinted and looked very close at the grainy intracellular contents. Dead cells floated about, unadhered to the bottom of the plate and brighter. The cells of the new culture looked a little smaller and seemed to be vibrating (although this was likely just the microscope itself).
2. No time point and no dilution produced a confluent monolayer. This is because our starting culture was too dilute and our incubation time was too limited. Perhaps if we could “grow up” the culture in several consecutive rounds we could have produced a confluent monolayer.
3. There is no evidence of contamination. No fungi, bacteria nor hair.
4. Humidity prevents the media from evaporated, and CO₂ is necessary for maintaining pH of the cell cultures.

Experiment 8B

1. The inducer could be an intracellular parasite that halts protein synthesis, thus prohibiting GFP expression. Alternatively, it could be too dilute or require certain conditions to become pathogenic.
2. The results in the handout are consistent — unfortunately, our positive control did not work.
3. No, our group did not detect any contamination.
4. E. coli is an extracellular pathogen and thus would elicit an extracellular response. For example, recruiting more macrophages. M. smeg is an intracellular pathogen and thus would elicit an intracellular response, such as apoptosis.

Experiment 8C

1. LPS and E. coli induced GFP.
2. LPS and E. coli were about equal, while M. smeg did not induce GFP.
3. The FACS results are not particularly novel for M. smeg — it is likely just an older culture that could not infect the macrophages. Alternatively, the strain of macrophages used might not be less susceptible than other macrophages to M. smeg infection. Overall, there was more likely something irregular with the M. smeg (most probable), conditions (also propable) or macrophages (somewhat probable) to explain the lack of GFP induction by M. smeg.
4. By concentration, a single microgram of LPS and fifty microliters of E. coli induced the strongest response. However, there was a signicant drop in induction with a hundred microliters of E. coli — this could be due to either macrophages dying from E. coli exposure, or possibly faulty lab technique. a deeper knowledge of interaction between E. coil and macrophages is necessary to confirm which concentration of E. coli gave the strongest response. After all, a hundred microliters of E. coli might induce the strongest and most widespread GFP expression initially, followed by a sharp drop-off as the cells die.

Part I

M1 peaks represent live cells without GFP expression (based on negative controls0 and M2 peaks represent live cells with GFP expression. E. coli shows a dose response at 100µl, but I would prefer to repeat the experiment to disprove experimental error since the M2 peaks for lower concentrations are about equal. For M. smegmatis is is impossible to tell whether dose response occurs, since there is only a single FACS diagram available to describe its induction of GFP expression. The lack of GFP induction by M. smeg is surprising because it is an intracellular pathogen. However, it is possible that the strain used lacked virulence factors or that it somehow halted host protein synthesis.

Part II

Sample A depicts T cells from the thymus. My reasoning is that there are lots of CD4+CD8+ cells and a few others scattered throughout; this majority of double-positive cells indicates is consistent with what I would expect from cells isolated in the thymus. Sample C depicts splenic T cells. My reasoning is that there are lots of CD4-CD8- cells (either non-thymocytes and/or dead T cells) and a few helper as well as cytotoxic T cells. A majority of double-negative cells is consistent with what I would expect when isolating T cells from the spleen.