Monday, March 26, 2012

A well studied marker impress me

    Hello folks. In the last one week I have been thinking of many different topics to post. There is so much in the world of microbiology to select. So, I thought why not post something on immunology. After all, Medical Microbiology significantly attributes much to elegance of Immunology.

   Pentraxins are a superfamily of molecules, well conserved over the evolutionary process, containing a very characteristic pentraxin domain. All the members of this family share an 8 aalong conserved sequence (HxCxS/TWxS, in which x is any amino acid) in the pentraxin domain, called pentraxin signature.

Fig 1: Amino acid sequence of pentraxin members (Source)

     Based on the primary structure of the subunit, the pentraxins are divided into two groups
  1. Short Pentraxins. E.g.: C-reactive protein (CRP) and serum amyloid P-component (SAP) 
  2. Long Pentraxins. E.g.: Pentraxin 3 (PTX3) 

Fig 2: Three-dimensional structure of human C - reactive protein (CRP). (Link)

        CRP was the first purified pentraxin, which was named after its ability to bind in a calcium-dependent fashion the C-polysaccharide of Streptococcus pneumoniae. Short pentraxins are often involved in acute phase reactions in a Ca2+ dependent manner and produced by the hepatic tissue. 

          The long pentraxin prototype is PTX3. It is produced in a variety of tissues and is involved with inflammation. PTX3 interacts with several ligands, including growth factors, extracellular matrix component and selected pathogens, playing a role in complement activation and facilitating pathogen recognition by phagocytes. This is made possible by interacting with specific pattern recognition molecules.

Fig 3: Pentraxin in humoral immunity

    I  bet u heard of Atherosclerosis. In simple terms, it is a condition where the inner parts of arterial blood vessels (which carry oxygenated blood to various parts of body), forms plaque and reduces the lumen diameter. This often leads to various conditions. (For details click here)

       So, if we could detect this perhaps in earlier stages, we could improve the life quality of the victim. For this, we need a marker. CRP was often used as a marker but with little significance. CRP is an acute phase protein, and levels fluctuate in many conditions. Moreover, the CRP levels are diagnostic only at later stages. This desperately needs a better marker. Almost for more than 4 years now, PTX3 was considered as a possible marker and much was research was done (Refer Savchenko A etal)

          In a recently published study in PloS one by Michael Knoflach, it was found that the PTX3 is independently associated with atherosclerosis and manifest cardiovascular disease but not early vessel pathology.

          For die hard fans of microbiology, i would like to make an additional statement. Pentraxin3 is a better marker of dengue infection better than C-reactive protein in dengue (link)

Further reading:

1. Cecilia Garlanda. Pentraxins at the crossroads between innate immunity, inflammation, matrix deposition, and female fertility. Annual Review of Immunology Vol. 23: 337-366. (Link)

2. Barbara Bottazzi, An Integrated View of Humoral Innate Immunity: Pentraxins as a Paradigm. Annual Review of Immunology Vol. 28: 157-183 (Link)

Tuesday, March 20, 2012

Flush the hidden HIV out….

    HIV (Human immunodeficiency virus) is the one virus, which has dominated the microbiology research over the years. No matter, how good a drug you design or a combination of it, the virus always has a way to get out of it.

Writing the entire biology of HIV is impossible as there is too much amount of data available in the literature. So let me try to concise. HIV is a retrovirus member belonging to Lentivirus group. The virus roughly spherical in shape with a cone shaped core possessing 2 single stranded RNA of Positive sense. The T lymphocytes that possess CD4 are the target for the HIV. In addition to CD4 receptor, CXCR4 or CCR5 serve as the co receptor.

Fig 1: Genetic structural organization of HIV. (Click here for source)
       Despite a + sense RNA the virus doesn’t follow the usual route of making all the components in cytoplasm and escape in the form of progeny. It instead makes a proviral DNA that integrates itself into the genome. Here again, unlike most of the viruses, HIV doesn’t wait for the cell to divide (The cells which divide, during mitosis provides an open nucleus, which gives the virus a chance to mix into our nucleus). Instead, the virus has the capability to pass through the nuclear pore. This enables the virus to infect quiescent cells also. (That’s the damn good and important feature of HIV in comparison to other retrovirus). The exact mechanism, how the virus enters the nucleus is still under study. Many components are implicated such as the LEDGF, Integrase, MA protein, Central DNA flap, capsid etc etc. (Click here for more details by Michael bukrinsky, Jeremy Luban, Yamashita etal 1, 2)

        Now that you understand what the HIV does, u can understand what the problem is. If you know that the anti HIV drugs works well only when the virus is active my job of saying more is saved. (If I could say in a more generalized way, a drug works on the target, only when the target is active). So you see the immediate problem. What about those HIV which are latent, inside a cell. This is exactly what happens. No matter how good your antiviral is, you get an elimination of upto 99% (as in HAART), but the one’s hiding there are not eliminated.

So, how do you treat these patients??

         There are two options. The first is to keep treating. You give antiretroviral therapy and continue to give so that every time the HIV is lurking out, the ART attacks. The protocol induces selection pressure and soon the resistance emerges.

         How about trying to flush the hidden HIV? That’s what exactly David Margolis team did. The clinical study conducted by Margolis tried to flush out the HIV thus providing the second method stated above. It is too early to start talking about this. However, that’s definitely worth news. (For link click here).

         Before I go further, let me give you a bit of information on Vorinostat (suberoylanilide hydroxamic acid). It is a lab hydroxamic acid derivative intended for anti-neoplastic activity. A second generation polar-planar compound, it binds to the catalytic domain of the histone deacetylases (HDACs). Vorinostat crosses the blood-brain barrier.

Fig 2: Structure of Vorinostat (chemically N-hydroxy-N'-phenyloctanediamide) (Click here for details)

        Bear with me, a note on HDAC also. HDAC is a very important enzyme almost ubiquitously found inside nucleus to carry out normal process. A NF-κB p50–HDAC1 complex is shown to constitutively bind the latent HIV LTR and induce histone deacetylation and repressive changes in chromatin structure of the HIV LTR, causing changes that impair recruitment of RNA polymerase II and transcriptional initiation. (Click here for source).
Fig 3: Action of vorinostat
So, what did the study team do?

         They selected six stable HIV infected men who were on ART. They administered Vorinostat to them and within hours the RNA expression of HIV was induced in CD4 cells. Quote from science news “evidence that the virus was being forced out of its hiding place”. So far so good, but this is where I want to interfere.

        The coming out of RNA definitely is good, as the virus is now out of hiding and available for target. But, it’s the RNA that’s out. The master DNA is still in. So I really wonder if we have pushed the hiding HIV out or woken up the sleeping HIV. (Don’t consider me a negative critique. As far as I know the results are not yet published, as on March 20, 2012. Once the article is out I guess I have answers to many of my questions).

My take home message is simple…“We now know how to get the HIV out of its latent stage”.

Monday, March 12, 2012

Microbiophobia- Bubble blown out

         I have been writing for some time on the pathological inflictions of the microbe on the humans. It doesn’t matter if you are a medical professional or not, you are concerned about your health. The more knowledge we acquire at the molecular level, more research papers claiming the nastiness of the microbe, and the more public news paper highlighting some epidemics or pandemics, people are scared to core. Many have come up with this concept of “Microbiophobia”. Oh yeah! I know a very vague and probably very dry, sterile topic to talk about in this blog. Yet, let me try to give u a bird’s view of this. 

          Before I just began inscribing I typed this word in google search box “Microbiophobia”. And to my surprise, there was “14800 results”. Well, that indicates to me how serious people are about this. 

         Imagine a new born never exposed to any microbe. To maintain him the same would be an exceptional task. He can eat only autoclaved food, live inside a microbe free room not allowed to mix with anybody, nobody even cuddles him. Oh! That baby would just not have a normal life. And even after such extra ordinary measures the baby would be weak and may not be scoring well immunologically. Such a test has not been done on humans, but some of the poor mice did have to. The reason why I’m starting with human baby as a hypothetical assumption is that we are most caring about the newborn, where we try the maximum to avoid any microbial contact. Sure, that makes sense, because a new born isn’t equipped well with an immune system to combat. 

          But if the parent insist it and reduce the exposure limits it could be really dangerous. We are equipped with a very smart immune system. But the smartness is mostly adaptive. This means the immune system has to be trained to identify pathogens and their components. A person who has a normal flora will do this job, by constantly challenging the immune system and keeping it busy. If you wouldn’t give your immune system a chance then it will not react when there is a real need. Certain people are too cautious of Microbial flora. They insist on too much sterility of materials they use. And their immune system is just too weak. (On the other hand there are people who are just too dirty and the immune system is unable to handle). It’s the science giving wrong information? No, I don’t believe so. Science says the right thing but what many understand is the wrong side of it (“What I said is right, but what you understood is wrong”). Science says that some microbes are pathogenic and people understand that microbes are bad (They simply forgot to digest the “Some”!!). What I mean to impress in you is that only some are bad, not all. (Jump to this link to have a neat idea on good vs. bad bugs).

Photo: Germ free animals (Click here for source)

          The microbes which are present in our body as normal commensal are involved in various functions such as keeping the immune system active, resisting the invading bacteria etc which are well known. It also serves some less known functions such as attractiveness to mosquito (Refer Niels O. Verhulst etal), susceptibility to auto immune diseases (click here), and brain functioning (click here). So better keep your microbiota in good shape.

So now I turn the question back to the readers. Is super hygiene a good practice?

          My plain answer is not. It is enough if you are relatively hygienic. It has been shown that children who play with soil are less asthmatic compared to children who are bred in very hygiene (Am sorry I don’t remember the reference were I read it, but believe me). Another common example is use of hands to eat rather than using a well sterilized spoon. The normal skin flora is much better. 

          I’m saying all this just to impress a single point. Microbiophobia is an increasing problem (come on, we already have enough phobia’s to deal with). Too frequent use of disinfectants in day to day life is not indicated. If you are Health care worker, this doesn’t apply because the environment is more likely to contain dangerous organism in higher concentration. Your infrequent hygiene may affect you or your patients and may lead to Hospital Acquired infections. If there’s a child in your home and want to play outside in ground let him. Don’t torture him with all the coverings possible and smear him with all the disinfectants you know. Once he finishes playing, give him a shower so as not to overload the system. 

Too much or too less, both the ends are the problems.

Further reading:

1. Getting the Dirt On Immunity: Scientists Show Evidence for Hygiene Hypothesis (Link).

Monday, March 05, 2012

Normal intracellular viral immunity- A problem in gene therapy?

Have you ever thought of this question…? “When a virus enters the cell the cell has to identify that there’s an intruder. Given the fact that the cell itself has a genetic material in it, the cell has to differentiate between foreign and self genetic material. So how does the cell do it? What is the implication when using viral vectors to deliver genes?”

The safest bet would be with DNA virus and the dsRNA virus. A normal cell would not have a DNA fragment (Am talking with reference to eukaryotic cell such as that in we humans), or rarely a dsRNA fragment. The dsRNA in normal cases will be taken care by host cells via pathways like RNAi pathway. (I will discuss about it in some other post. For a few basics, click here). The single stranded RNA would be more difficult to assess as we have similar types in our cells. However they have sufficient differences to be recognized.
Fig 1: TLR and RIG-I two antiviral innate immunity pathways.

So when a virus enters the cell the signals are detected by TRIF (TIR-domain-containing adapter-inducing interferon-β), MyD88 (Myeloid differentiation primary response protein 88) and RIG-I (Retinoic acid inducible gene I, also known as DEAD-box protein 58). For an excellent review article on the mechanism please refer to the review by Rashu B Seth et al. Newer signals and responders are identified, an active field of research. 

No matter how the pathway starts, the signaling cascade culminates in MAVS. The mitochondrial anti-viral signaling protein (MAVS), or otherwise known as CARDIF, IPS-1, KIAA1271 and VISA, is mitochondria associated protein that regulates type I interferon through a well structured and highly regulated production of NF-kappaB and IRF3. The N-terminal CARD domain of MAVS interacts with RIGI.

Fig 2: DNA Sensors and Anti‑Viral Immune Responses.

The CARD domain stands for Caspase recruitment domain. Caspases are enzymes that are involved in Apoptosis regulation in cells.

Genetic diseases are a group of inborn errors that can clinically vary depending on the gene affected. Loss of a gene function can be corrected by injecting the cell with a functional DNA and incorporating it into the genome (Gene therapy). Who else can deliver a gene to the cell better than a virus in natural conditions? There are many ways to achieve a gene therapy procedure. Major protocols under study include 
  • Cationic liposomes 
  • DNA condensation and receptor mediated gene transfer 
  • Viral vectors such as Retrovirus, Adenovirus, Adeno-associated virus etc 

Fig 3: AAV life cycle, Shyam daya etal
Adeno-associated viruses are small single-stranded DNA viruses of the Dependovirus genus belonging to Parvoviridae. AAV-2 is used in experimental studies. AAV-2 enters the cell through an endosomal route, is transported to the nucleus by motor proteins, and enters the nucleus, everything happening in a flash.          
So now that you have understood the background from various angles, let me put things together. This blog post is based on the paper, “Innate responses to AAV vectors” by Rogers et al. This paper looks at the hindering effect of innate immune response to the AAV vectors which has the potential to deliver important genes.

The paper starts with an introduction to the AAV importance as a vector in gene transfer and also some current perspectives of the innate immunity to virus in a cell. The sensors detect the arrival of an extracellular gene leading to multiple phosphorylation event (This is common. Most of the intracellular signaling is based on phosphorylation or de-phosphorylation of various intermediates). The pathway culminates in stimulation of NF-κB Classical pathways.

Fig 4: A proposed model for innate immune
recognition of AAV vectors
The second part of the paper focusses on the Specific response to a AAV. Here it was noted that the ssAAV (Single stranded AAV) typically produced less immune response compared to other Adenoviral vectors, and the scAAV (self-complementary AAV) produced more response in comparison to the ssAAV.

A model was also proposed to show the possible mechanism of innate immune recognition of AAV vectors.        

The last part of the paper is dedicated to its implication in clinical practice.

The AAV is a very good vector of choice for treating genetic diseases as it provides a flexibility at various levels. The problem is that the viral matter can elicit a immune response, which often is low, but good enough to have some tissue degradation. That may lead to treatment failure. So the possibility of use should be supported by some antagonists of these response.
Further Reading:

Rogers etal. Innate responses to AAV vectors. Frontiers in Microbiology. September 2011; Volume 2 Article 194