T cells and immunosuppression after organ transplantation

In the 7630 course transplantation unit, it has been mentioned that graft rejection is majorly a T cell unintentional wrongdoing and immunosuppression regimen should be able to take care of that. What comes to be a little surprising is dating from mid 50's uptill now, though we have learned the mechanism of how rejection happens but we still don't have a surefire way of saying right away what kind of success we will have with a given transplantation. Reason to that is the transplantation success more relies on post procedure patient monitoring and the right cocktail of immunosuppressive drugs (least toxic but effective) tailored for an individual with multiple given physical conditions. Post-transplantation, for some time the focus is on the transplanted organ, its health and how its coping with the host body, but later on the long term overall patient survival becomes the critical point. Why? because with organ transplantation, its not only the transplanted organ, but the all of host's body at stake. The major reason to this is the inherent toxicity that long term immunosuppressive drug regimen ultimately manifests. I believe the holy grail in organ-transplantation is to find better biomarkers which give the patient's state of the system picture accurately, timely, preferably non-invasively and the immunosuppression, such that its localized, does what its supposed to and nothing else. Of course if in future we are able to make organs in-vitro that are made with patient's own cells, then we don't have to worry about immunosuppression (ref: tissue/organ skeleton re-population by stem cells and "chromosome 6" by Robin cook). But for now, ideal long term immunosuppression may be the biggest challenge in transplantation and that's where T-cells come in picture.

Standard immunosuppression therapy has two phases, the more aggressive, "stand down or I WILL shoot" approach during the initial post-transplantation period. It is to prevent acute rejection episode with cocktail of multiple drugs targeting multiple aspects of host immune system. Then later on, as the patient and the graft stabilize a bit, a subdued but still effective dosing of choice immunosuppressants specifically targeting T-cells, is administered such that toxicity is minimal. Before describing more on T-cells, immunosuppression mechanism and resultant toxicity, a short description of different immunosuppressive drug classes is in order. These are mainly glucocorticoids, anti inflammatory drugs, antimetabolites, antibodies and lymphocyte cell-proliferation inhibitors via interaction with immunophilins. Glucocorticoids are administered generally in the first phase and suppress cell mediated immunity (pro-inflammatory genes of interleukins and TNF ). Anti inflammatory properties of a drug suppress the attraction of immune system components to a site of inflammation / injury. This means less epithelial adhesion, emigration, chemotaxis, phagocytosis, metabolic acceleration and release of inflammatory mediators. Antimetabolites and cytostatics are targeted to suppress cell division specifically that of T and B cells. For example purine and pyramidine analogs stunt the DNA replication process thereby inhibiting cell division. Polyclonal and monoclonal antibodies are directed towards T and B lymphocytes, some cause lysis of these cells by complement and others cause opsonization, some target receptors such as IL-2 and CD-3 to prevent interaction and activation. As you can imagine these drugs can be given in combinations minding the patient specific and sound just wonderful for the treatment. However none of these drugs are without serious adverse impacts if not given correctly. The same goes for immunosuppressants which kick in the first phase of the therapy and stay on for the long term suppression. I am indicating towards drugs that interact with immunophilins to inhibit T cell proliferation. This is the drug class that is T cell specific and has major implications in long term immunosuppression drug regimen and drug toxicity due to lengthy exposure.

I shall further delve into the last drug class, their partial pharmacodynamics, T-cell behavior under these drugs and as an example nephrotoxicity vs chronic rejection in my next post. I hope you found this post useful.

Thanks !

- Rahul

Mind/Body Connection

There has always been a kind of "folk knowledge" that there is a connection between the mind and body. This idea has made some headway in the skeptical scientific community with proof of the placebo effect, and there are an increasing number of studies being done to see how the nervous system interacts with other systems in the body, like the immune system, for instance. This is just one of the articles I found really interesting showing the connection between the nervous and immune systems, which I found on ScienceDaily.com. (neat site!) I think the take-home message from all these studies is that everything is much more connected than we previously thought, and I look forward to learning the cellular specifics of how the nervous system interacts with other systems in the body, even tho that the interaction itself exists is something people have intuitively known for years.

ScienceDaily (Nov. 19, 2001) — In a discovery that demonstrates a clear link between the mind and body at a molecular level, scientists have shown that a chemical signal which normally allows nerve cells to communicate with each other –to alter sleep cycles, for example -- can also re-direct actions of the immune system.

---

The research in mice confirms mounting evidence from studies of cultured cells that the nervous system directly influences the immune system. It has prompted new experiments to determine if the nerve-generated signal or its receptors in the immune system might make good drug targets to control asthma or allergies.

“This is the first clue of a practical pharmacological approach to using the nervous system for both improving immune defenses and damping harmful immune responses at their roots in diseases as diverse as arthritis and asthma,” said Edward Goetzl, MD, professor of medicine and immunology at the University of California, San Francisco.

Goetzl is lead author on a scientific paper on the research in the November 20 issue of the Proceedings of the National Academy of Sciences. The work is a collaboration between UCSF and the University of Edinburgh. Goetzl is also senior author on a companion paper on the research in FASEB Journal. (FASEB stands for the Federation of the American Societies for Experimental Biology.)

The finding is based on experiments with “knockout” mice whose immune cells can’t receive the normal neuropeptide signal known as vasoactive intestinal peptide, or VIP.

In the nervous system, VIP normally stimulates nerve cell signaling and survival, and regulates neural biological clocks. The scientists found that VIP also affects the migration of the immune system’s T cells and T cell secretion of protein signals for other immune cells, both of which are central to the body’s normal defense against infection. Through its action on T cells, VIP can affect the process in which the immune system turns against the body, such as in asthma and arthritis.

In the PNAS paper and in the companion paper in the FASEB Journal, the researchers showed that the strength of the VIP signal received by the T cells regulates the balance between two types of immune T cells, Th1 and Th2. Th1 is normally involved with protection from bacterial invasion and other defenses, but Th1 in excess can lead to autoimmune disorders. Th2 protects from parasitic infections and autoimmunity, but in excess can lead to allergies.

The researchers discovered the effect of VIP on the Th1/Th2 balance by examining the relative production of the Th cells’ protein products, known as cytokines. When the balance is tipped toward Th1 in knockout mice lacking a critical form of a VIP receptor, allergy is suppressed and resistance to some types of infections is boosted, along with other reactions, they found.

The research did not determine if the impact of the neuropeptide VIP is sufficient to change the course of infections, inflammation or autoimmune disease in which T cells are involved.

The researchers caution that VIP has such broad effects on immune function that blocking its action with drugs might risk triggering one kind of immune malady while it relieves another. However, the new findings clearly demonstrate the potential of neuroregulation of T cell functions and suggest the potential value of developing VIP-like drugs with greater immune selection than VIP itself, Goetzl added.

Senior author on the PNAS paper is Anthony Harmar, PhD, professor of neurosciences at University of Edinburgh. Co-authors are post-doctoral fellows Julia K Voice, PhD, and Glenn Dorsam, PhD, in the UCSF medicine and immunology departments; and Yvonne Kong, research assistant in the same departments. Also on the study are post-doctoral fellows Sanbing Shen, PhD; Katrine M. West, PhD; and Christine F. Morrison, PhD, all at University of Edinburgh.

The research was funded by the National Institutes of Health and the Medical Research Council of the United Kingdom.

---

Adapted from materials provided by University Of California - San Francisco.

Reference cited:
University Of California - San Francisco (2001, November 19). Signals From Nervous System Influence Immune System, Study Shows. ScienceDaily. Retrieved October 22, 2009, from http://www.sciencedaily.com­ /releases/2001/11/011116064459.htm

HIV Drug Classes

My lab does a lot of research on the pharmacology of HIV drugs, so for those interested, I thought I would follow up our class discussion on drug classes with a few examples of the drugs in each class (generic names) and various pros and cons.

NRTI (Nucleos(t)ide Reverse Transriptase Inhibitors)
Emtricitabine, Lamivudine, Zidovudine, Zidovudine/Lamivudine (Combivir), Didanosine, Tenofovir, Tenofovir/Emtricitabine (Truvada), Stavudine, Abacavir
Target: Blocking HIV RNA transcription to dsDNA by competitively binding to the viral DNA and terminating chain extension.
Pros: Drug binding halts the proliferation of viral DNA, slowing the progression of the virus. If the drugs are at a sufficiently high level intracellularly before infection, can have a prophylactic effect and prevent the initial infection.
Cons: Drug levels must saturate the amount of viral RNA to fully prevent proliferation, otherwise the unimpeded RNA strands could still cause infection. Also, NRTIs have demonstrated mitochondrial toxicity.

NNRTI (Non-Nucleoside Reverse Transcriptase Inhibitors)
Efavirenz, Nevirapine, Etravirine, Delavirdine
Target: Changes conformation of reverse transcriptase, decreasing the activity of the enzyme’s catalytic sites.
Pros: Drug targets the enzyme, instead of the enzyme’s product, which diminishes the possibility of getting fully or partially formed dsDNA.
Cons: As with NRTIs, the drug levels must saturate the enzyme to fully prevent viral proliferation.

PI (Protease Inhibitors)
Indinavir, Ritonavir, Lopinavir/Ritonavir, Atazanavir, Nelfinavir, Tipranavir, Indinavir
Target: Inhibits the viral protease enzyme which cleaves the viral polypeptide coded for by the dsDNA.
Pros: Can be used in all retroviruses. Prevents the dsDNA from being cleaved into its more virulent components which prevents disease proliferation.
Cons: Must be used in combination with other antiretrovirals (usually NRTIs) in order to have full efficacy.

Integrase Inhibitors
Raltegravir
Target: Inhibits integrase, the enzyme which inserts viral DNA into host DNA.
Pros: Can be used to treat multiple retroviruses, not just HIV, including mutated HIV virus. Early results show very good efficacy. Also used in combination with other drug classes to have greater effect in preventing viral proliferation.
Cons: Still early in the ballgame to determine long term effects of the drug. May be best suited as an additional drug in HAART. Prevents last stage of viral addition to host DNA, which may not be the best drug target, as the viral DNA will have already been produced before infection is halted.

HBV Surface Markers

Ok, so I felt like my last post (on HBIg) was fairly unclear regarding the markers of hepatitis B infection. Hopefully this clears things up.

 

The basis of hepatitis B infection (HBV) is the hepatitis B surface antigen (HBsAg). It is immunogenic, but not infectious. HBsAg is the protein in the viral envelope, and antibodies to surface antigen is what is measured for in a simple HBV test.

 

Ongoing HBV viral replication is indicated by the presence of serologic markers of the virion core. The virion core is composed of HBeAg and HBcAg. HBeAg is consistent with ongoing viral synthesis, and is generally found in areas with high viral endemicity. Strangely, when antibodies to HBeAg are present (Anti-HBe) this indicates relatively low infectivity in the individual, and may be a marker of the integrated stage of infection (Dienstag 2008).

 

HBcAg (Anti-HBc) is indicative of ongoing, chronic liver disease.

 

Lower titers of IGg anti-HBc with anti-HBs, show that infection with HBV had occurred in the past. High titers of IGg anti-HBc with low levels of anti-HBs show that viral infection has occurred, and is still present (Sherlock 1990).

 

 

References Cited

 

Dienstag, Jules L.

2008            Hepatitis B Virus Infection. New England Journal of Medicine 359(14):1486-1503.

Sherlock, S.

            1990            Hepatitis B: The Disease. Vaccine 8(Supplement):S6-S10.

 

Reactive Arthritis

Reactive arthritis (ReA) is a non‑purulent joint inflammation that usually follows bacterial gastrointenstinal or urogenital infections. The classic presentation of ReA is characterized by an asymmetric arthritis usually in the lower limbs associated with urethritis, conjunctivitis and occurrence of other articular or extra‑articular manifestations. ReA is classified as a type of seronegative spondyloarthopathy.
ReA not only affects the joints, but is also a systemic disease with extra‑articular symptoms. The clinical picture is dominated by syndromes of enthesitis (enthesopaties), peripheral arthritis (acute or subacute oligoarthritis mainly of the lower limb joints), pelvic and axial syndromes (spinal involvement with sacroilitis) and extramusculoskeletal syndromes.

Approximately 65–85% of patients with ReA are HLA‑B27 positive. Regardless of the preceding infection, the clinical picture is similar, but management can differ according to the triggering infection. Treatment of Chlamydia‑induced ReA should be started with antibiotics because of several mechanisms by which Chlamydia can cause persistent infection. The disease may have an acute or self‑limited course, however some patients develop chronic arthritis.

Etiology and pathophysiology of Reactive Arthritis:
Several factors contribute to the development of ReA, including:
1- The presence of bacteria or bacterial products in the joint and the local immune response directed against these bacteria
2- The effect of the arthritogenic peptides derived from ReA – triggering bacteria (Salmonella and Yersinia), which epitopes are presented by phagocytes to cytotoxic T lymphocytes in synovial membrane
3- Recognition of bacterial antigens outside
the cells, and forming complexes with class I human leukocyte antigens (HLA) and class II histocompatibility antigen (MHC II) presented on CD4 + and CD8 + T cells in Chlamydia infection
4- Tendency to persistent bacterial infections
and imbalance between tumor necrosis factor α (TNF‑α), interferon‑γ, IL‑12, IL‑10 activity (increased IL‑10 levels in the intestines, urogenital and respiratory system in patients with persistent infections).
5-Genetic factors also play an important role
in the etiology of ReA. The HLA‑B27 antigen is found in 65–80% of patients with ReA.

Extra‑articular manifestations of Reactive Arthritis:

Enthesopatic syndrome:
Heel pain, Achilles tendonitis, pain at the tibial tubercle
Pelvic and axial syndrome:
Sacroiliitis, spondylitis
Eye:
Conjunctivitis, iritis, keratitis, episcleritis
Genitourinary:
Urethritis, prostatitis, hemorrhagic cystitis, cervicitis
Gastrointestinal:
Diarrhea
Skin:
Keratoderma blennorhagica, circinate balanitis, oral ulcers, hyperkeratotis nails, erythema nodosum
Cardiovascular system:
Aortic disease, EKG conduction abnormalities
Renal:
Proteinuria, microhematuria, aseptic pyuria

Investigation of Progressive Inflammatory Neuropathy among Swine Slaughterhouse Workers – Minnesota, 2007—2008

This article appeared in MMWR on Feb. 8, 2008.

The Minnesota Department of Health conducted an investigation to characterize the outbreak of unexplained neurologic illnesses among workers in a swine slaughterhouse in southeast Minnesota. As of January 28.2008 12 workers at this plant had been identified with progressive inflammatory neuropathy (PIN). Median age of the 12 workers was 31years, and six patients were female. All 12 patients that had PIN reported being healthy before the onset of neurologic symptoms.

The symptoms of PIN reported by workers ranged from acute paralysis to gradually progressive symmetric weakness over periods ranging from 8 to 213 days. Eleven patients had evidence of axonal or demyelinating peripheral neuropathy by electrodiagnostic testing. A sample of cerebrospinal fluid was obtained from seven patients, and they all showed elevated protein levels with no or minimal pleocytosis. Five of these patients had evidence of inflammation on a spinal MRI (4 patients in peripheral nerves or roots and one patient in the anterior spinal cord).

All 12 patients with PIN reported either working at or having regular contact with an area known as the warm room where swine heads were processed (head table). Thus, a case-control study was conducted among the plant’s workers to identify the specific risk factors associated with neurologic illness. Ten patients out of the 12 initial reports of PIN were chosen that had confirmed or probable PIN. Also two stratified control groups were formed: 1) a random selection of 48 healthy warm-room workers and 2) all 65 healthy head-table workers. Blood samples and throat swabs were collected from all consenting case patients and controls. As of Jan 30, 2008 the lab investigations had not identified any infectious agent from the blood and throat swab specimens that would explain the occurrence of PIN.

The results of the case-control study highlighted several important aspects that could contribute to PIN among the workers. Seven of the ten case-patients were significantly more likely to have worked at the head table than warm-room controls (12 of 48). Also, case-patients were more likely to have removed brains or remaining skeletal muscle from the pig head (4 of 7) than head-table controls (8 of 65). Importantly, the illness was not determined to be related to previous travel outside or within the US, nor related to exposure to chemicals, fertilizers, insecticides, or use of medications and receipt of previous vaccinations.

An environmental assessment of the plant was conducted the better understand the methods of processing as well as the personal protective gear used by the workers. The investigators found that a compressed air device was used in the plant to harvest brain tissue from pig heads at the head table. The compressed air disrupted the brain material into a liquefied form that made it easier to remove the brain. However, this technique caused splatter possibly including aerosolized brain material to which the workers operating the device and other nearby might have been exposed. In response to this investigation, the plant suspended harvesting of brains and instituted additional protective gear that included face shields and long sleeves.

To date, three plants in the US used compressed air to remove pig brains including one in Nebraska and one in Indiana. Nebraska plant did not have reports of Pin, but the Indiana plant had similar histories of PIN as in Minnesota for workers exposed to head processing activities at that slaughterhouse. As a result of this, all three plants have stopped using compressed air devices for brain removal.

The FDA inspected the pigs processed by these plants and no food risk was identified for the general population. This investigation in Minnesota suggests that in the process of using the compressed air device, workers might have been exposed to aerosolized brain material through inhalation or contact with mucous membranes. The current hypothesis for development of PIN in the swine plant is that worker exposure to the aerosolized pig neural protein might have induced an autoimmune-mediated peripheral neuropathy. Thus, is it just a cross-reaction in the immune system caused by the similarity of the pig neural proteins to human proteins?

Using rare codon pairs to attenuate viruses.

A friend of mine who works at the CDC turned me on to a new technique they're working on to construct safe attenuated viruses to use as vaccines: Genome wide changes in codon pair bias.

A quick review of codons: each of the twenty amino acids is encoded by at least one 3 letter codon in RNA: for example, GCC is translated by ribosomes into an Alanine. Because there are 4 nucleotides availible, there are 4^3, or 64 possible 3 letter codons. Since only 21 (20 AA + a stop codon) are actually required, many amino acids are encoded by multiple sets of codons (eg. UCU, UCA, UCG, and UCC all encode Serine) The human genome, however, preferentially uses certian codons (GCC is four times as prevelent as GCG, though both encode Ala), and pairs of codons (GCAGAG and GCCGAA encode the same two amino acids, but the former is 7 times as prevelent in the human genome). As it turns out, our cellular machinery is tuned to translate common codon pairs more effecitvely than rare codon pairs.

A group of researchers has developed a way to build long DNA sequences, and with those sequences, generate poliovirus de novo, without any template. Using this technique, they were able to develop equivalent versions of poliovirus with identical protiens, but encoded with a varying degree of rare codons and rare codon pairs.

Because our cellular machinery translates rare codons poorly, a very large number of rare codons renders the virus totally ineffective at infection - creating no immune response. By including some rare codons, but some standard codons, the researchers were able to create a virus that is effective enough to trigger an immune response (and subsequent immunity - remember, the virus has identical protiens), but weak enough to prevent a true viral infection - an effectively attenuated virus.

As you may remember, some current live, attenuated vaccines have the possibility of reverting to virulence: for example, the oral polio vaccine, which has only 5 attenuating mutations, can, on rare occasions, revert to a virulent form - which is why it is no longer available on the US market. A rare codon virus, on the other hand, has hundreds of mutations, each contributing, in a small way, towards attenuating the virus; this leaves no chance of the virus reverting to virulence. This technique also allows for the systematic weakening of a virus - potentially allowing vaccine developers to precisely adjust the strength of an attenuated virus to maximize immunogenic effectiveness.

Reference:
J. Robert Coleman, Dimitris Papamichail, Steven Skiena, Bruce Futcher, Eckard Wimmer, and Steffen Mueller
Science 27 June 2008 320: 1784-1787 [DOI: 10.1126/science.1155761] (in Reports)

Smoking, Sugar Uptake, and Their Roles in Crohn’s Disease...

Smoking is a risk factor and a contributor to many diseases. Scientists have discovered that smoking tends to worsen the effects of Crohn’s disease. The aetiology and pathogenesis of Crohn’s disease, however, is still unknown. The mechanism of the effect of smoking on the colon is also unknown. Pathogens might cause some changes in bowel motility and susceptibility but it is hard to predict the mechanism of those changes. Moreover, smoking may produce complex changes in immune function including reduced natural killer cell activity in peripheral blood leukocytes and decreased immunoglobulin concentration in the serum and saliva.


Furthermore, some studies have shown that patients with Crohn’s disease consume more refined sugar than normal healthy people. Other studies revealed that people with Crohn’s disease who consume more sugar in their diet show exacerbated symptoms compared to those who consume less sugar. Since nobody knows how sugar injures the intestine, doctors recommend their patients to eliminate sugar from their diet, or change their diet from a high-carbohydrate to a low-carbohydrate.


Now, we know that sugar and smoking are bad for patients with Crohn’s disease. The challenging question is what will happen if a patient with Crohn’s disease has a combination of both risk factors? Some studies showed that sugar intake and smoking were independent risk factors for Crohn’s disease, but a combined exposure did not result in a further increase of the risk. Some researchers think that these factors might operate through a common mechanism. I did my research trying to get an answer but i found nothing. I believe those people who figured out that there is no increase in risk if you combine both factors should work hard to figure out the mechanism behind this.

Hepatitis B Immunoglobulin

Immunocompetent patients who are already positive for hepatitis B (HBV) are treated with INF-µ. Patients who have had a recent exposure should immediately be treated with hepatitis B immunoglobulin (HBIg). Interestingly, HBIg may have a variety of other applications for HBV infected patients. For example, it can be  administered to infants born to HBV positive mothers within 24 hours of birth to prevent neonate infection.

 

Perhaps the most interesting application, is that of the case of post-transplant patients. Immunosuppressed transplant patients may be treated with INF-µ2b to prevent allograft re infection. However, if allograft reinfection does occur, INF-µ2b can cause rejection, as was the case in a 1997 clinical trial of the drug. Discouragingly, if patients who are transplanted for chronic active infections do not receive any kind of post-transplant antiviral therapy, average estimated survival times range from only 1 – 5 years.

 

Treatment with HBIg in immunosuppressed post-transplant patients has been remarkably successful. HBIg has been shown to prevent allograft reinfection by maintaining anti-HBs titers at a level >250IU/L. This is so successful that it even prevents reinfection in patients with actively replicating viral infections!

 

Though this treatment is extremely effective, it is also expensive. For the first year of HBIg treatment, patients may be looking at a cost around $38,000! For every year after that, patients can expect to pay around $15,000 annually.  It seems extremely unfair that such effective treatment is reserved only for those who are able to afford it…

Got IBD? Sit under the Bodhi tree.

There is an intimate connection between the immune system and the nervous system. This makes sense because both play a role in systemic homeostasis. For example inflammation can be triggered by CNS stimulation. An electro-chemical signal starts in the CNS and is propagated into the periphery through sensory C-fibers. These nerves release substance P which cause the release of histamine from mast cells. Chemicals, physical touch, and emotional stress can all trigger the release of substance P. Beyond the hormonal connection there may be a direct synaptic connection. There is evidence of a Neuro-Immune synapse. These NI synapses are between axon terminals and immunocompetent cells such as mast cells, T-cells, and macrophages. These synapses are still under investigation but they appear to participate in cross talk between the nervous system and the immune system. So if your stress’n out find a quiet place and get your zen on, your body will appreciate it.
-Randy
P.S. Below is a pretty cool article if your into that sort of thing.

Citation:
Nakanishi M, FurunoT. Molecular basis of neuroimmune interaction in an in
vitro coculture approach. Cell Mol Immunol. 2008Aug;5(4):249-59.