Question

A tuberculin skin test involves injection of a purified TB (Mycobacterium tuberculosis) antigenic protein under the skin. A positive skin test is a local swelling and redness at the site after 48 to 72 hours.

(a) What cell type is responsible for this reaction?

(b) Why do people who have been vaccinated with BCG (attenuated bovine tuberculosis bacterium) sometimes have a positive skin test even if they do not have active tuberculosis?

Rapamycin (sirolimus, Rapamune) is a common anti-rejection drug given to organ transplant patients, usually for the rest of their lives after transplant.

(a) What is the target of rapamycin on cells of the immune system and what cells are affected?

(b) Why does this effect cause immunosuppression?

(c) Why does the drug have to be given for the lifetime of the patient?

Studies have been done on the use of anti-CD40 ligand antibody ruplizumab (trade name Antova) in the autoimmune disease systemic lupus erythematosis (SLE, Lupus). Lupus is characterized by B cells making anti-DNA autoantibodies.

(a) According to the naming conventions, what does the name of this drug tell you about its structure? (b) What type of immune cells would it bind to?

(c) Why do you think that it was tried in the treatment of lupus?

Answer 1

a) T-cells sensitized by prior infection are recruited to the skin site where they release lymphokines. These lymphokines induce induration through local vasodilatation, edema, fibrin deposition, and recruitment of other inflammatory cells to the area.

b)If you were vaccinated with BCG, you may have a positive reaction to a TB skin test . This reaction may be due to infection with the TB bacteria . However, in some people, BCG may cause a positive skin test when they are not infected with TB bacteria . A positive reaction is more likely to mean you have been infected with TB bacteria if: • You recently spent time with a person who has TB disease; or • You are from an area of the world where TB disease is very common (such as most countries in Latin America and the Caribbean, Africa, Asia, Eastern Europe, and Russia); or • You spend time where TB disease is common (homeless shelters, migrant farm camps, drug-treatment centers, health care clinics, jails, prisons) . Unlike the TB skin test, TB blood tests are not affected by prior BCG vaccination . The TB blood tests are less likely to give a false-positive result in people who have received BCG .

Rapamycin

a)The mechanistic (previously referred to as mammalian) Target Of Rapamycin (mTOR) is a serine/threonine protein kinase that is inhibited by rapamycin, a compound produced by bacteria originally isolated from the soil of Easter Island that inhibits the proliferation of eukaryotic cells .

The mTOR protein kinase is found in two discrete complexes, mTOR complex 1 (mTORC1) and mTORC2, each of which contains distinct protein components and phosphorylates different substrates. mTORC1 is acutely inhibited by rapamycin, which has been used therapeutically and as a probe to gain insight into mTORC1 regulation and function. mTORC2 is significantly less sensitive to rapamycin and is only inhibited by chronic treatment in certain cell types and tissues. As a result of its relative insensitivity to rapamycin, the physiological function and molecular targets of mTORC2 have been harder to decipher.

mTOR in the liver

The role of mTOR in the liver has been investigated over the past several years, primarily using three different genetic mouse models: mice lacking hepatic TSC1 (L-TSC1KO), which have hyperactive mTORC1 signaling; mice lacking hepatic Raptor, with decreased mTORC1 signaling (L-RapKO); and mice lacking hepatic Rictor (L-RicKO), with decreased mTORC2 signaling.

b)Rapa's main mode of action is the inhibition of cytokine-activated signal transduction, but clearly, any inhibitory effect on proinflammatory cytokine transcription (however minor) would complement its immunosuppressive efficacy. The underlying mechanism for this is not known but may stem from the inhibition of costimulatory pathway transduction as this has been implicated in the transcriptional activation of several cytokine genes.

c)

. Rapamycin, a drug originally found in Easter Island bacteria, is another. It can lengthen the lives of old mice by 9 to 14 per cent, and it boosts longevity in flies and yeast too.

But rapamycin has its downsides. For a start, it strongly suppresses the immune system. That is why it is currently given to people who receive new organs, to stop them from rejecting their transplants. Rapamycin can also increase the risk of diabetes. In mice, rats and humans, the drug weakens the ability to stabilise levels of sugar in the blood. Individuals who take it for a long time become resistant to insulin, and intolerant to sugar.

You’d expect the opposite. Longer-lived animals ought to be better at dealing with sugar, and less likely to suffer from insulin resistance. Indeed, that’s what you see in individuals that cut down on calories.

Dudley Lamming from MIT has discovered a possible reason for its two-faced streak. Rapamycin blocks a protein called TOR (which, fittingly, stands for “target of rapamycin”). TOR was the first known protein that influences longevity in all of the four species that scientists commonly use to study ageing: yeast, worms, flies and mice.

TOR runs in two different gangs, known as mTORC1 and mTORC2. In each one, it joins forces with a separate group of proteins. Lamming found that these collectives (biologists call them complexes) do different jobs, and rapamycin exerts different effects by targeting each one. By disrupting mTORC1, it extends lifespan. By disrupting mTORC2, it messes up our ability to process sugar.

Lamming made his discovery by depriving TOR of two of its associates: Raptor, a protein found in mTORC1, and Rictor, a protein found in mTORC2. He could knock out either protein at will, in the livers of specially engineered mice.

When he reduced levels of Rictor, and thus the mTORC2 complex, the rodents’ livers started producing extra sugar and they developed a severe intolerance to the stuff. A dose of rapamycin didn’t worsen any of these problems.

On the other hand, if Lamming deprived the mice of Raptor, and thus mTORC1, the mice were able to process sugar in the normal way. However, rapamycin no longer extended their lives.

ruplizumab

Ruplizumab is an anti-CD40L monoclonal antibody. The binding of CD40 to its ligand, CD40L, is a critical element in T cell activation. In systemic lupus erythematosus, CD40L is over-expressed on T cells, B cells, and monocytes.

The autoimmune response is executed via cognate interactions between effector immune cells and antigen presenting cells. Cognate interactions provide the immune effectors with specific signals generated through the antigen receptor as well as with second, non-specific signals, generated from the interaction of pairs of cell-surface molecules (costimulatory molecules) present on their plasma membrane. Disruption of this second, non-specific costimulatory signal results in the interruption of the productive (auto)immune response, leading to anergy, a state of immune unresponsiveness. The CD28:B7 families of molecules and the CD40:CD40L pair of molecules are considered as critical costimulatory elements. Disruption of the CD28:B7 interaction using a genetically engineered soluble form of the inhibitory molecule CTLA4 in vitro, as well as in experimental models of rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), led to the inhibition of the autoimmune response. Similarly, promising data stem from the use of an anti-CD40L monoclonal antibody (mAb) in murine SLE. While such treatments prevent the development of autoimmunity in animal models, this preventive approach is inapplicable to human diseases. However, the rational bench-to-bedside approach led investigators to clinical trials of CTLA4-Ig and of two different humanized anti-human CD40L mAbs in patients with RA and SLE, respectively. Initial experience with the use of CTLA4-Ig in patients with RA is encouraging, since in one short-term trial the construct was well-tolerated and produced clinically meaningful improvement of the disease in a significant proportion of those treated. Surprisingly, the anti-CD40L mAb treatment approach in human lupus was not fruitful, since short-term administration of the anti-CD40L mAb ruplizumab in lupus nephritis was correlated with life-threatening prothrombotic activity despite initial encouraging data in the serology and renal function of the patients. Also, IDEC-131 anti-CD40L mAb treatment did not prove to be clinically effective in human SLE, despite being well tolerated. Precise tailoring of the administration schemes for these novel therapeutic modalities is awaited.Finally, conceptually different approaches to block costimulation by inhibiting the induced expression of costimulatory molecules or the transmission of their specific intracytoplasmic signal have already produced encouraging preliminary results.