Idiotypic Vaccines for Lymphoma

Lymphomas are a heterogeneous group of malignancies arising in the lymphoid tissue. Lymphomas often start in the lymphatic system in locations such as the lymph nodes. They may then spread to other locations such as the lungs, liver or bone marrow. 25-40% of lymphomas arise at specific extranodal sites, such as splenic marginal zone B-cell lymphomas that arise in the spleen (Marcus R. and Sweetenham J. W., 2007 ) Most Lymphomas are B cell in origin, with a minority being T- cell. There are two main categories of lymphoma: Hodgkin’s Lymphoma (HL) and Non- Hodgkins Lymphoma (NHL) (Freedman J., 2005). Hodgkin’s lymphoma is a lymphoma composed of Reed-Sternberg cells surrounded by reactive non-neoplastic chronic inflammatory cells (variable numbers of lymphocytes, plasma cells, eosinophils, histocytes and neutrophils) and variable degrees of fibrosis.

Lymphoma accounts for over 3 % of cancer occurring worldwide. 75,000 people in the UK are living with lymphoma and over 13,500 people are diagnosed with lymphoma every year. The most common are B cell cancers diffuse large B cell lymphoma and Follicular lymphoma (FL) (Freedman J., 2005).

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Idiotypic Vaccines for Lymphoma

When Animation Meets Science...

Multimedia artists and animators work primarily in the movie industry, computer and video games, advertising, and computer systems design services. They both draw by hand and use computers to create the series of pictures that form the animated images or special effects seen in movies, television programs, and computer games. In 1962, Computer engineers developed a method of drawing both straight and curved lines using computers and since then it has allowed for rapid advances and ultimately allowed people to create even the most technical of illustrations on a computer.

Today technical illustration is used in many different ways; it is used by engineers or scientists to communicate highly complex specifications with their peers as well to communicate with people outside their specialisations, and also by highly skilled specialists to communicate with the general public. Many multimedia artists model objects in three dimensions by computer. Some artists, usually animators, work with programmers to make their three-dimensional models move. They can transform complex information into striking images that stimulate imagination, facilitate learning, and record scientific discovery.

A medical illustration is a visual representation that is the result of art skills expressed in a tangible or virtual medium that conveys medical or biological information. It is collaboration between the biologist and the animator that does the magic. Over the past two years, the partnership has resulted in eye-catching images and animations of Biomolecules that grace scientist presentation slides, their lab's website etc. Medical or Scientific animators are borrowing tools from Hollywood to breathe life into cells and molecules on screen.

Scientific animators work with software similar to that used to create special effects and animated films in Hollywood, including a program called Autodesk Maya. But instead of creating monsters and explosions, they pull in data from a variety of sources, including review and research papers, to bring molecules and cells to life on screen. Dozens of papers can be necessary to inform a single animation. Animators also tap into scientific databases, such as the Protein Data Bank (http://www.pdb.org), to extract molecular structures, images, microscopy data and other key information. Some animators also spend a lot of time in close discussions with scientists to nail down details of how a molecule or cell moves or interacts with others.


From the human genome to the latest robotic surgical technique, the demand for accurate, effective medical illustration is continuously expanding. Medical illustrations and animations appear in virtually all media and markets used to disseminate medical, biological and related information. Attorneys use medical illustration to clarify complex medical information for judges and juries in personal injury and medical malpractice cases.

While medical illustrations are still widely and consistently used for print and exhibits, the trend is toward greater use in multimedia and interactive designs, particularly those involving animation. Many, but not all, medical illustrators also work in three dimensions, creating sculptured anatomical teaching models and museum exhibits, models for simulated medical procedures and prosthetic parts for patients.

In academia, animators also develop courses and adapt animation tools for use in science. Eventually, they hope, visualizations could become a research tool used to develop, test and refine biomedical hypotheses, not just a method of communication. “As we get more data, we need better ways to synthesize the data into models,” says Dr. Iwasa. Dr.Iwasa is in the vanguard of scientific animators working in academia. Harvard's cell-biology department hired her three years ago to facilitate communication among faculty members and other scientists. Since then, she has worked with about a dozen researchers to visualize the molecules, pathways and cellular processes that they study, such as cell death. She also has a growing freelance business, 'one-micron illustration', creating animations, illustrations and websites for researchers at Harvard and other institutions. You can get a glimpse of her animation on https://iwasa.hms.harvard.edu/.

Academia is only a small part of the market for scientific visualizations. Scientific animators can also be found at a growing number of commercial animation and design studios that specialize in biomedical work. Medical-device, biotech and pharmaceutical companies use animations about their latest products in sales, marketing and educational materials. Visualizations also end up in museum exhibitions, classroom teaching tools, digital textbooks and documentaries, and on journal covers and websites.

Because of the variety of assignments medical illustrators typically experience, they should be accomplished in a wide range of art methods and media production skills to meet the current needs of the Bio-communication industry. These methods and skills range from advanced drawing, painting and sculpture techniques in tangible media, to functional concepts and techniques involved in the production of commercial and graphic art, to up-to-date computer graphic skills in still and motion media.

Content and anatomical accuracy is paramount in the field of scientific illustration; images are designed and created to communicate specific content. Therefore, it is most rewarding for detail-oriented individuals who genuinely enjoy and have natural ability in both art and science. Having a background in art or an eye for design and visual storytelling is crucial for scientific animators. An innate sense of aesthetics or some basic training in lighting, colour and composition to enable visual expression through drawing or other media is key to success, says Graham Johnson, an animator who will soon be starting a position at the University of California, San Francisco. He will be continuing his work developing software that, for example, integrates molecular modelling tools with animation programs to better connect raw scientific data with animation capabilities.

A strong foundation in general, biological and medical science is also necessary to enable the illustrator to fully comprehend and then conceptualize complex biological and medical information. Subjects range from structures in the real world that can be directly observed to the theoretical and unseen, such as molecular processes. Highly developed visualization skills to transform such complex information into two-dimensional and three-dimensional images that communicate to diverse audiences are essential. Those interested in scientific illustration should enjoy working alone and in teams during problem-solving, and be able to work closely with clients to understand not only the project itself but the client's often unspoken needs as well. Writing, research and ancillary computer skills are also valuable.

Scientific animators around the world are seeing rising demand for their work from sectors including academic research, publishing, biotechnology and the drug industry. Driving this interest is an expansion in digital media connected with devices such as the iPad, and a burgeoning appreciation from publishers, scientists, educators, museum staff and others of the power of three-dimensional (3D) visualizations to communicate complex concepts. Animation studios have proliferated in the past five years, and medical-illustration master's-degree programmes have expanded their class sizes, with graduates generally able to find jobs with animation firms and research institutions. More and more scientists are seeking out animators, and a few, hoping to tinker with animation to aid their research rather than build a fully fledged career in it, are learning to use the tools themselves.It is rightly said by Dr.McGill, Director of Molecular Visualization at Harvard Medical School, “When you're going through the process of making visualization, you come up with new questions and open up new ways of thinking.”

Suicide Gene Therapy against HIV :Thymidine Kinase gene

Acquired immunodeficiency syndrome (AIDS) and its associated disorders are caused by human immunodeficiency virus (HIV). HIV exhibits a tropism for CD4+ T lymphocytes, which constitute the primary target for HIV infection in vivo. Initial infections appear to be latent and are characterized by an extended, asymptomatic stage of pathogenesis. Disease progression results in a state of prolific viral replication that eventually leads to the massive depletion of CD4+ T lymphocytes that occurs during AIDS.  Even though substantial progress has been made in the molecular characterization of HIV, therapeutic treatment of HIV mediated pathogenesis has proved difficult to attain, particularly due to uncertainties in understanding mechanisms related to the persistence of viral latency.

Resting T lymphocytes are nonpermissive for HIV replication; even then the virus efficiently binds to the CD4 receptor and is internalized.  From this latent state, the virus can be initiated into productive infection by factors that activate quiescent CD4+ lymphocytes into cellular proliferation. It appears that the latent provirus is activated by the same inducible cellular transcription factors that promote T-cell proliferation upon presentation of the appropriate antigen. This feature of HIV replication raises therapeutic possibilities; agents that are toxic to activated HIV-infected T cells would be expected to inhibit HIV replication and the ensuing pathogenesis. 

HIV-infected T cells upon activation, initiate a program of viral gene expression that is stringently controlled by two nuclear regulatory proteins coding viral genes tat and rev. The main function of tat gene product, Tat, is transcriptional activation from the viral 5' long terminal repeat (LTR) promoter by binding to structured RNA target sequence, the transactivation response element (TAR). The action of the rev gene product, Rev, is posttranscriptional; Rev selectively induces the nuclear export of a constitutively expressed pool of structural-gene mRNAs that contain a cis-acting sequence of extensive secondary structure, the Rev-responsive element (RRE). The viral cis-acting sequences TAR and RRE, whose regulatory activity is tightly controlled by Tat and Rev, respectively, afford an excellent means for the targeted expression of cytotoxic agents in HIV-infected cells that express these regulatory proteins.
 
Suicide Gene therapy is a technique for modifying the cellular genome for rendering cells sensitive to chemotherapeutics or toxins by introducing “suicide genes”. Here conditionally cytotoxic human herpes virus type- 6 thymidine kinase gene (HHV-6 TK) is used as suicide gene in Tat expressing HIV infected cells.  HSV-1 TK expression is not deleterious to mammalian cells, but it can, unlike mammalian thymidine kinase, selectively phosphorylate certain nucleoside analogs such as acyclovir and ganciclovir (GCV) to their monophosphate..  Ganciclovir(GCV),  is phosphorylated first by the viral thymidine kinase to nucleoside monophosphate (GCV-MP) and then by cell kinases to yield the triphospho form of the drug(GCV-TP).Gancyclovir triphosphate  when  incorporated into DNA, leads to inhibition of DNA synthesis .This TK-GCV system induces accumulation of p53 and increases cell surface expression of death receptors  leading to apoptosis involving the Fas-associated death domain protein (FADD) and caspases. One more advantage of the HSV-TK-GCV system is the bystander killing effect whereby HSV-TK positive cells exposed to GCV are lethal to surrounding HSV-TK negative cells via transport of GCV-MP, GCV-BP, and GCV-TP trough gap junctions to adjacent cells.Thus using HIV-2 LTR as inducible promoter for the thymidine kinase gene ,the HIV infected cells can be specifically killed .Similar strategy were  used earlier for specific killing of  HIV infected cells  where  LTR promoter is used for selective expression of  pro-apoptotic Bax gene leading to apoptotic cell death of tat expressing cells ( McCoubrie JE. et al,2004) or  LTR- herpes simplex virus (HSV) virion host shutoff gene (vhs) construct where vhs  encodes a protein which nonspecifically accelerates the degradation of mRNA molecules, leading to inhibition of protein synthesis in HIV infected cells ( Hamouda T. et al,1997).

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Suicide Gene Therapy against HIV : Thymidine Kinase gene

Single Cell Analysis: Flourescence Microscopy

Since their advent, proteomics and genomics have developed in ways that underline the rate and volume of data acquisition and analysis. They have, by necessity, worked on large populations of cells and thereby reported on population averages rather than their distributions.In this process they have missed rare, but important events and been unable to analyse cells that are only produced in small numbers. This is of course not by choice but owing to a paucity of techniques that allow experimentalists to measure protein levels at the single-cell level.

In addition, genome sequence information provides powerful insights into cellular complexity but limited information pertaining to how individual parts of a cell are work together in time and space to form dynamic cellular processes and how cellular interactions consequently translate to create higher order functions. This currently hampers an understanding of the mechanisms behind the morphological design of organisms that depends on programmes of cellular division, apoptosis, immune response etc. that are closely linked to these spatially and temporally dependent signals.

Parameters based upon averages of large populations are often misleading. Cellular heterogeneity is widespread in bacteria and increasingly apparent in eukaryotic cells. The complex and highly interconnected network of signalling pathways, their spatially dependent nature and reliance upon low-abundance molecules produces stochastic behaviour that subsequently underpins heterogeneity in cellular systems. The noise in biological function subsequently expresses itself in many different forms, from noise-driven divergence of individual cell fates through to noise-induced amplification of signals.

Similarly, the study of rare cells such as stem cells and progenitor cells does not lend itself to high throughput population-based protocols. In these cases, the development of single-cell analysis techniques that allow multiple measurements to be conducted on the same cell as a function of time is vital if we are to unravel the inner workings of these extraordinary systems.

Focusing on the characteristics of a group can obscure the differences between the individuals in it. Yet when it comes to biological cells, scientists typically derive information about their behaviour, status, and health from the collective activity of thousands or millions of them. Analyzing individual cells allows researchers to distinguish between a uniform population of cells and a group of cells with members having, say, different protein content.

A more precise understanding of differences between individual cells could lead to better treatments for diseases such as cancer, diabetes etc. Detecting minute differences between individual cells could improve medical tests and treatments. An understanding of the relationship between biological heterogeneity and signalling pathway regulation that may result in disease states is therefore critical and offers the potential to drive novel therapeutic interventions developed in response to single-cell behaviours.

Thus, Single-cell studies are crucial in order to productively study the complexity of intracellular processes.

However, tools that are capable of harvesting large amounts of proteomic data from single cells remain rather limited, largely owing to the difficulty involved in dealing with the small volumes and quantities of analytes concerned. Despite the limitations, over the last decade or two, there has been significant progress in developing assays capable of determining levels of specific proteins and interrogating enzyme activity in single cells .Fluorescence Microscopy is one such method which can be sucessfully used for Single cell analysis.

Lots of agents are used for the fluorescence, one such widely used protein is EGFP (Enhanced Green Fluorescent Protein). In the 1960s and 1970s, GFP, was first purified from Aequorea victoria and its properties studied by Osamu Shimomura. In A. victoria, GFP fluorescence occurs when aequorin interacts with Ca2+ ions, inducing a blue glow. Some of this luminescent energy is transferred to the GFP, shifting the overall color towards green. However, it’s utility as a tool for molecular biologists did not begin to be realized until 1992 when Douglas Prasher reported the cloning and nucleotide sequence of wtGFP in Gene. A 37 °C folding efficiency (F64L) point mutant to this scaffold yields enhanced GFP (EGFP) which was discovered in 1995 by the lab of Ole Thastrup. EGFP allowed the practical use of GFPs in mammalian cells. EGFP has an extinction coefficient (denoted ε) of 55,000 M−1cm−1. The fluorescence quantum yield (QY) of EGFP is 0.60. The relative brightness, expressed as ε•QY, is 33,000 M−1cm−1.

EGFP expressing cells under Flourescence Microscope

EGFP can also be expressed in different structures enabling morphological distinction. In our cases, the gene for the production of EGFP is spliced into the genome of the organism in the region of the DNA that codes for the target proteins and that is controlled by the same regulatory sequence; that is, the gene's regulatory sequence now controls the production of EGFP, in addition to the tagged protein. In cells where the gene is expressed, and the tagged proteins are produced, EGFP is produced at the same time. Thus, only those cells in which the tagged gene is expressed, or the target proteins are produced, will fluoresce when observed under fluorescence microscopy. The fluorescence can be used to even identify position of the target protein and the its amount produced at a specific time point.

Analysis of such time lapse movies has redefined the understanding of many biological processes in our case helped us to understand protein transport, and RNA dynamics, which in the past had been studied using fixed (i.e., dead) material.

Quick Facts of IL-1R

• Interleukin-1 receptor (IL-1R) is a cytokine receptor which binds interleukin 1 (IL-1) which is a cytokine.

• There are two types of IL-1 receptor, termed type I and type II receptors, each with three extracellular immunoglobulin (Ig)-like domains but limited sequence similarity (28%) and different pharmacological characteristics.

• IL-1R contains three Ig domains and, together with the highly homologous IL-1R accessory protein (IL-1RAcP), forms a receptor complex for IL-1 alpha, IL-1 beta  and IL−1 receptor antagonist (IL-1RA). Of the three Ig-like domains, domains 1 and 2 are tightly linked, while domain 3 is completely separate and connected by a flexible linker.

• Both the receptors exist in transmembrane (TM) and soluble forms: the soluble IL-1 receptor is thought to be post-translationally derived from cleavage of the extracellular portion of the membrane receptors.

• Both of them bind to the Interleukin-1 alpha, interleukin-1 beta and Interleukin-1RA which are cytokines that participate in the regulation of immune responses, inflammatory reactions, and haematopoiesis. Interleukin-1 (IL-1) exerts pleiotropic effects on a variety of tissues through binding to its receptor. interleukin-1 (IL-1R) play an important role in innate immunity by regulating the activity of distinct transcription factors such as nuclear factor-kappaB(NF-kappaB).

• IL1RB is the beta subunit of the receptor and is known also as IL1R2. The human gene encoding the beta subunit was cloned by McMahan et al (1991). In the nomenclature of CD antigens, this receptor has been given the designation CD121b.

• IL1R2 is structurally incapable of signalling and functions as a decoy receptor, binding and inhibiting the effect of IL1. IL1R2 can reduce the number of signalling complexes consisting of IL1R1, IL1RAcP, and IL1, by binding to IL1RAcP and thus has the ability to modulate the responsiveness of cells to IL1 . The type I receptor is primarily responsible for transmitting the inflammatory effects of IL-1 while type II receptors may act as a suppressor of IL-1 activity by competing for IL-1 binding.

• Also opposing the effects of IL-1 is the IL-1RA.The IL-1RA binds IL1 and transduces IL1 signals in cooperation with the co-receptor IL1RAcP to initiate an inflammatory response. In the nomenclature of CD antigens, this receptor has been given the designation CD121a. The human gene encoding the alpha subunit was been cloned by Sims et al (1989).

• All the IL-1 receptors appear to be well conserved in evolution, and map to the same chromosomal location.

• Cells may produce soluble variants of the IL1 receptors (designated sIL1R1, sIL1R2), which can compete with membrane-bound receptors for ligand binding. They can act as molecular sinks and scavengers for free ligand and can play a regulatory role by modulating ligand availability.

• IL-1 Family Receptors:

The IL-1R family consists of members that are characterized by the presence of extracellular immunoglobulin-like (Ig) domains and by an intracellular TIR domain.They are:

-IL-1ra/IL-1F3
-IL-1 Rrp2/IL-1 R6
-IL-1 RI
-IL-18 R alpha/IL-1 R5
-IL-1 RII
-IL-18 R beta/IL-1 R7
-IL-1 RAcP/IL-1 R3
-SIGIRR
-IL-1 RAPL1/IL-1 R8
-ST2/IL-1 R4
-IL-1 RAPL2/IL-1 R9

• The IL-18 receptor (IL-18R), following binding to IL-18, forms a complex with IL-18RAcP to initiate downstream signalling.

• IL1 receptor-related protein-2 (IL-1Rrp2) is the receptor for the agonists IL-1F6, IL-1F8, and IL-1F9, which also uses IL-1RAcP as a second chain. Thus, IL-1RAcP appears to be promiscuous since, in addition to IL-1RI and IL-1Rrp2, it also associates with ST2, which has recently been shown to bind IL-33.

• IL-1R2 and SIGIRR are two inhibitory receptors, the former lacks the Toll/IL-1 receptor (TIR)- domain whereas the latter contains a single Ig domain for the extracellular segment.

• The only members that still remain without any identified function in this subfamily are IL-1RAPL and TIGIRR.

• The adaptor molecules are: the Myeloid differentiation factor 88 (MyD88) and the MyD88-adaptor-like (MAL, also known as TIRAP).

• TIRAP is the TIR domain-containing adaptor protein inducing interferon-alpha.

• MyD88 has a modular structure consisting of an N-terminal death domain (DD) separated by a short linker region from the C-terminal TIR domain.

• The IL-1 receptors and TLRs (Toll-like Receptors) share a common TIR motif in their cytoplasmic domain. While IL-1 receptors respond to traditional IL-1 family ligands, the TLRs elicit inflammatory responses via the recognition of pathogen-associated molecular patterns (PAMPs) such as bacterial proteins, nucleic acids, and cell wall components.

• The TIR domain is responsible for the propagation of the signal within the cell through interaction with a complex signalling cascade. The presence of an intracellular TIR domain is the hallmark of TLR/IL-1R super family consisting of IL-1R subfamily and the TIR-domain-containing adaptor proteins.

• The IL-1 Ligands & IL-1R/TLR Family plays critical roles in inflammation and host defence. These molecules play important roles in both innate and adaptive immunity.

• MyD88 is an important signalling adaptor for both TLR and IL-1R family members. Resident skin cells utilize IL-1R/MyD88 signalling to promote neutrophil recruitment.

• A novel IL-1RI co-receptor, TILRR, links with the signalling receptor complex and enhances recruitment of the MyD88 adapter, controls induction of the Ras GTPase, and amplifies activation of NF-kappaB and inflammatory genes.

• One of the other adaptors involved are TRIF–related adaptor molecule (TRAM) where TRIF is TIR-domain-containing adapter-inducing interferon-β. TRAM contains a TIR domain in the C-terminal region and functions exclusively in the TLR4 pathway. TRAM interacts with TIR-domain-containing adapter-inducing interferon-β (TRIF) and the sterile alpha- and armadillo-motif containing protein (SARM). These adaptors bridge the TLR/IL-1R receptors to the intracellular molecules that transduce their signals into a biological response and play a central role in innate immunity.

• SARM contains a TIR domain at C-terminus, two “sterile a” motif (SAM) protein-protein interactions domains, and an Armadillo repeat motif (ARM).It functions as an inhibitor of TRIF-dependent TLR signalling.

• Another adaptor like toll-interleukin 1 receptor (TIR) domain containing adaptor protein (TIRAP) is required to act as a bridge for MyD88 in TLR2 and TLR4 signalling, while TRIF is used in TLR3 signalling and, in association with TRAM, in TLR4 signalling.

• Based on the type of adaptor molecules involved, the TLR/IL-1R-induced pathways can be sub-grouped in two classes: MyD88-dependent and MyD88-independent responses.

• In the MyD88-dependent pathway, MyD88 associates with IL-1R-associated kinases like IRAK4, IRAK1 and/or IRAK2. IRAK4 in turn phosphorylates IRAK1 and IRAK2 and promotes their association with TNF receptor associated factor-6 (TRAF6), which serves as a platform to recruit the TGF-beta activated kinase-1 (TAK1).

• Once activated, TAK1 activates the IKK complex, composed of IKK alpha, IKK beta , and NEMO (IKK gamma), which catalyzes phosphorylation and subsequent degradation of IkappaB rendering NF-kappaB free to translocate from the cytosol to the nucleus and activate NF-kappaB-dependent genes.

• TLR/IL-1R receptors associate with MyD88 through homotypic interactions between their respective TIR domains. This interaction then allows MyD88 to recruit members of the interleukin-1 receptor-associated kinase (IRAK) family (IRAK1, IRAK2, and IRAK4) through homotypic interactions between their respective Death Domains (DDs).

• The MyD88-dependent TLR/IL-1R signalling plays a vital role early in life, but becomes less important for survival during ageing. This is likely consequent to activation and/or maturation of TLR-independent innate immunity. Moreover, these findings seem to suggest that innate immunity is more important upon the very first encounter with a pathogen. Once adaptive immunity is generated, however, resistance to infection becomes quite efficient even in the absence of crucial functional components of TLR signalling.

• Once activated by their respective ligands, IL-1R, IL-18R, and TLRs engage with one or more adaptor proteins. These adaptors, namely, MyD88, MAL/TIRAP, TRIF, and TRAM are recruited, in various combinations, to the cytoplasmic domains of the receptors through homophilic interactions between Toll/IL-1 receptor (TIR) domains present in each receptor and each adaptor.

• TLR, IL-1R and TNF-R signalling to NF-kappaB converge on a common IkappaB kinase complex that phosphorylates the NF-kappaB inhibitory protein IkappaB.

• An aberrant activation of TLR/IL-1R signalling can promote the onset of inflammatory and autoimmune diseases. The development of therapeutic strategies for the control of their function have potential to cure inflammatory and immune disorders such as sepsis syndrome, asthma, atherosclerosis, Alzheimer’s disease, rheumatoid arthritis (RA).

• Pharmaceutical modulation of TLR/IL-1R signalling pathways by inhibitors, such as decoy peptides and synthetic mimetics that interfere with protein-protein interactions between signalling molecules of the TLR/IL-1R super family, might yield clinical benefits in the treatment of inflammatory and autoimmune diseases.

• The TIR domain of TLR/IL-1R proteins is a putatively suitable target. In particular, the BB-loop region may be regarded as a critical functional interface of TIR domain for its critical role in proper signalling. BB-Loop Decoy Peptides are short amino acid sequences of a protein that are expected to mimic its interaction surface and to prevent interaction of the prototype proteins with their partners.

• For e.g. A TIRAP decoy peptide consisting of the 14 amino acid-long sequence in the BB-loop (LQLRDAAPGGAIVS), fused to the Drosophila antennapedia homeodomain to facilitate the intracellular delivery, specifically blocked TLR4-induced activation of NF-kappaB without affecting the TIRAP-independent TLR9 response. In vivo administration of TIRAP inhibitory peptide counteracted the lung inflammatory response in healthy C57BL/6 mice.

• BB-loop heptapeptides derived from MyD88 and IL-18R inhibited homomeric interaction of MyD88 TIR domain or full-length MyD88 in vitro e.g. cell permeable derivative of the MyD88 BB-loop decoy heptapeptide (RDVLPGT).

• Toshchakov and colleagues performed systematic investigations of decoy cell permeable peptides containing TIR domain BB-loop sequences derived from the adaptor proteins MyD88, TIRAP, TRAM, and TRIF as well as the receptors TLR1, 2, 4, and 6. These decoy peptides were all able to inhibit, with varying activity, the TLR signalling pathways.

• Bartfai and colleagues, by focusing on TIR-domain interactions between IL-1RI and MyD88, synthesized a low-molecular-weight molecule mimetic, hydrocinnamoyl-L-valyl pyrrolidine.

• One of the most effective compounds, termed ST2825, inhibited homomeric interaction of MyD88 TIR domains. This effect was specific for TIR domains and did not affect interaction of MyD88 DD.

Although much emphasis has been placed on the development of NF-kappaB inhibitors, generic inhibition of NF-kappaB may lead to undesired side effects. Hence, a challenging objective is to develop drugs like above that block its effects in specific pathways, while leaving its physiological functions in other contexts largely intact. A promising research is going on in this line ...So watch out for this space...

Gandhi and Me

What is your understanding of Mahatma Gandhi and his message in contemporary global scenario?

A debate on the relevancy of Gandhi in today’s time has become a routine affair on every Mahatma Gandhi Jayanti. Actually it should be other way round- Can we face the challenge of Gandhi's ideals and ideas? For some people Gandhi’s teaching is still relevant today whereas some think that India & the world at large has changed so drastically that there is no role of Gandhi in it. No matter what exactly the truth is, the biggest truth of all is, there is some kind of charisma in him which simply fails to fade. The great Scientist Albert Einstein once said about Gandhi: “I believe that Gandhi’s views were the most enlightened of all the political men in our time. We should strive to do things in his spirit: not to use violence in fighting for our cause, but by non-participation in anything you believe is evil”. The more I read about Gandhi, the more I become humble to the greatness of him who was seeking nothing for himself but was willing to die in order that others may live. The above sentiments are depicted rightly in his words “There are many causes that I am prepared to die for but no causes that I am prepared to kill for."

The name of Mahatma Gandhi, today, transcends the bounds of race, religion, and nation- states, and has emerged as the Prophetic Voice of the twenty-first century. Gandhi is remembered for his passionate adherence to the practice of Nonviolence and his supreme humanism. After the Great Buddha and Jesus, he once again demonstrated that Non-violence could also be an effective instrument of social change.

I think that Gandhi’s value can successfully demonstrate to a World, weary with wars and destruction that adherence to Truth and Non-violence is not meant for individual behaviour alone but can be applied in global affairs too. The science of war leads one to dictatorship, pure and simple. The science of non-violence alone can lead one to pure democracy. Power based on love is thousand times more effective and permanent than power derived from fear of punishment. It is a blasphemy to say non-violence can be practiced only by individuals and never by nations which are composed of individuals. The nearest approach to purest anarchy would be a democracy based on non-violence.

A society organised and run on the basis of complete non-violence should expect that even non-violence be practised in police force. Police ranks should be composed of believers in non-violence. The people will instinctively render them every help and through mutual cooperation they will easily deal with the ever decreasing disturbances. Violent quarrels between labour and capital and strikes will be few and far between in a non-violent state because the influence of the non-violent majority will be great as to respect the principle elements in society.
Similarly, there will be no room for communal disturbances. A non-violent army acts unlike armed men, as well in times of peace as in times of disturbances. Theirs will be the duty of bringing warring communities together, carrying peace propaganda, engaging in activities that would bring and keep them in touch with every single person in their parish or division. Such an army should be ready to cope with any emergency, and in order to still the frenzy of mobs should risk their lives in numbers sufficient for that purpose. Satyagraha (truth-force) brigades can be organised in every village and every block of buildings in the cities. If the non-violent society is attacked from without, there are two ways open to non-violence. To yield possession, but non-cooperate with the aggressor and prefer death to submission. The second way would be non-violent resistance by the people who have been trained in the non-violent way. A nation or group which has made non-violence its final policy cannot be subjected to slavery even by the atom bomb. The level of non-violence in that nation, if that even happily comes to pass, will naturally have risen so high as to command universal respect.

The adoption of satyagraha and nonviolent non-cooperation will oblige the elected political representatives to honestly execute the wishes of the poor and oppressed millions in the rural areas and effectively stop those programs which are not in the interests of the entire polity. In situations where the representatives do not act according to the wishes of the people, Gandhi said 'we can protest and teach people to resist and make it clear to the Government that it cannot look us in this manner.

India today faces the onslaught of multinational investment and globalization in every sector of its economy. Will the new economic policy relieve the hunger of or provide remunerative jobs to the millions living in India? I think the answer to this question is to reconstruct a more harmonious, poverty-free, nonviolent, and self-reliant society based on the ethical and moral principles with which Mahatma Gandhi experimented. Business gurus in India are talking about Mahatma Gandhi as new role model .The Father of the Nation is now being held up as the master strategist, an exemplary leader, and someone whose ideas and tactics corporate India can emulate. The key business thinkers are preaching how corporate India needs to revisit Gandhi's ideas and apply the lessons learnt from him to their leadership styles. Gandhi is being held as a 'strategist. I think that Gandhi’s ideas are of particular relevance to India at this juncture, as it struggles to find ways to inch closer to the 8-10 per cent gross domestic product growth rate necessary to become an economic superpower. Gandhi reinvented the rules of the game to deal with a situation where all the available existing methods had failed. He broke tradition. He understood that you cannot fight the British with force. So he decided to change the game in a fundamentally different way. He unleashed the power of ordinary people, inspired women and men in the country to fight under a unifying goal. Resource constraint did not bother him. He aimed at a common agenda: Poorna Swaraj (Complete Freedom). That was the motivation. Drawing lessons from this, India needs to fundamentally change the way it can grow.

Gandhi's leadership style is being termed as 'follower-centric' and one that took into account existing conditions before determining the strategy. Gandhi advocated having leadership styles that were dependent on the circumstances. When Gandhi was in South Africa, he launched his protests in a suit and a tie. But when he came back to India, he thought of khadi and launched non-violent protests on a greater scale. I believe that Gandhi's style of leadership as applied to corporate India would involve making even the lowest person in the organization believe in it and the significance of his contribution towards it. In business, empowerment is all about making sure everyone is connected to the organization's goals. Gandhi has a way of doing that: making sure that everyone in the cause is connected to the goal. An understanding of these qualities about him is what is happening now in corporate India. Apart from the implications of Gandhi’s values only in relation to the Business sector, the Gandhian vision is a means to rejuvenate the entire nation and all its other important sectors. For e.g., local initiatives can foster the production of goods in cottage industries for primary consumption based on the principles of need and consequently exchange. In such a positive and caring environment resolution of disputes through harmonious reconciliation of competing interests and appeals to the rich and elite to respond to their dharma is possible. These steps will recreate the fervour, which once ignited the nation, for authentic swaraj. If India can revert to rechanneling its massive human resources towards these endeavours through a commitment to the Gandhian path it can resolve its crisis. The poor women, children, and men of this nation can return to the Gandhian message of self-reliance. By forming local, self-governing, and democratic political organizations it is possible to non-violently resist oppression and exploitation both by the state and market forces.

Self-imposed curbs on one's wants and production for one's needs can be achieved by discipline and selflessness. Devoting oneself first to cultivate the land for basic nutrition and only secondarily for exchange value is a must. Realization that self-help and bread labour are useful methods to frontally attack the vicious circle of poverty is imperative. Creation of democratic cooperatives for managing and allocating human and non-human resources in the spirit of trusteeship is requisite.

Gandhi had a dream for India. Realizing this dream has become a nightmare. Today, we are living in a constant adjustment to changing conditions, which require a different kind of discipline. Now it rests on our shoulders, yours and mine to see that the democratic values in our country remains intact and that social justice, equity, gender equality is achieved for all .Rights should follow duties If we are able to achieve this, we shall be helping to reinstate Mahatma Gandhi's dream, I firmly believe it can be done. Gandhi's unfinished task is the biggest challenge before the youth.

Gandhi had great faith in the ultimate success of his mission, because he had infinite faith in the individual's capacity to change. He firmly held that the human nature is capable of radical reorientation; all one needs is a will to explore his own true self. This explains why Gandhi, all through his life was striving to take humanity on to the path of spiritual and moral growth. The progress of civilization, as it has evolved through the ages, id proof that human nature is a developing entity, capable of change for the better. The key to making Gandhi's vision a reality and practising his values is to persistently subvert selfish worldly pursuits with ethical livelihood. This is the immediate struggle every Indian, on an individual and collective basis, has to non-violently and passionately wage.

Mahatma Gandhi was one of the leading volunteers the country has ever produced. Have you ever done any volunteer work? If yes, please outline your efforts and the impact it may have made?

Nature provides. Not always though, especially when there seeps in interference from the human element. This is particularly true of the Melghat forest area in Amravati district of Maharashtra where, between 1992 and 1997, an estimated 5,000 children died due to malnutrition. When the media picked up this shocking story, it generated a wave of criticism directed at the government and its inability to provide for the tribals who have inhabited the forests for centuries. But that is where it ended. No solutions were found. There was nothing concrete in the wake of lip sympathy. Until a group of curious social workers from Pune decided to penetrate into Melghat on a fact-finding mission.
That led to the initiation of the setting up of “Melghat Mitra”, a group which works exclusively for the betterment of the Korkus, which is what the tribal community in Melghat is known as. Five years since it was established with no funds nor an NGO status, Melghat Mitra has emerged as the only body of volunteers who have notched up remarkable achievements in this area of 350 villages, most of which are totally cut off from civilisation. In the 3 monsoon months of 2009, Melghat Mitra ran a “Dhadak Mohim” program where nearly 250 volunteers from Maharashtra and elsewhere lived with the people in 12 villages over a period of 10 days.

I got the privilege to join the organisation as a volunteer in the first batch of July 2009. Besides helping the local government health machinery to deliver their services, we tried to understand the problems and lives of the Korku tribals in Melghat. This program gave us an insight into the complex and multi-faceted problem of malnutrition and helped us to look at the problem from different perspectives. I believe that malnutrition needs to be addressed at its roots by basically ensuring the sustainable livelihood mechanisms for the Korku families. In addition, access to basic health and education services will also have to be ensured. Our strategy was to develop the capacities of the people to solve their own problems rather than make them dependent on us. Therefore, we do not burden the people with our programs but help people in their own initiatives. We continue to raise our funds through the public, strongly believing that such an activity should run entirely through public support, not only in order to ensure accountability and transparency, but also to ensure that the problem of malnutrition in India is understood and owned by the people of our country. I believe that it is only with collective efforts of the common man and of every single citizen that we can address the deep-rooted poverty of our Korku friend’s and others in similar situation.

Our campaign was called by us as the 'We won't let a single child die campaign'. The children who died till now fall in UNICEF's grade 4 category of weight/age ratio, which indicates severe malnutrition. Many of them didn't even have the strength to stand on their own. According to the authorities, the children die because the parents do not take them to a doctor. When a child dies the authorities enter it in their records as a case of diarrhoea or measles. Truth, however, paints an entirely different picture. We have sited through our observation, many reasons for the prevalence of malnutrition in the area. Teenage mothers, large families, lack of medical care and superstition have led to these deaths. The Korku belief that childbirth will be difficult if a woman eats well during the last trimester has led to underweight babies. Besides, the pregnant women must return to work in the fields immediately after they give birth, and therefore cannot nurse their infants for long. The government dole of Rs. 800 to pregnant women has proved counter-productive. The idea of the dole is to limit the size of the family and provide the best possible care to the nursing mother and new-borns. However, most Korku women consider it an incentive to bear more children. Even a mother of 16 children is availing of the dole. Racked by ignorance and poverty, the Korku tribesmen are finding life a painful experience. The death of Melghat's children, infact, is a manifestation of a deeper malaise in the Korku tribe. A visitor to the area will marvel at the fact that the agricultural produce of Melghat is worth Rs 10 crore. But, the tribals have no security of food for the next day. Benami (Property law) holdings are huge and moneylenders hold sway. There is also a relationship between education and the problem of malnutrition. The Korkus do not send their children to school because they need more farm hands. So while boys along with their mother and father go to the farms and girls keep watch on their siblings. There was a primary school in our village run by the zilla parishad with the barest of teaching aids. Most of the schools here have only one room and one teacher, and all the children would sit in the same class to be taught by the same teacher. From standard IV, they would have to enrol in a residential ashram in nearby town, which is why there was a high dropout rate. Added to this was the fact that the inhospitability of the region did not attract too many teachers.I along with my group started abhyas wargas(study classes) and created a force of Boko Mitras (a child is called Boko in the Korku language and Mitra means Friends) who encourage children to attend classes, and ensure that learning is fun and not routine.

My experience in melghat was more than mere doing social work it was one of kind experience .The experience of living among the tribal people of India was journey of self-discovery. The Korkus there even though of their condition are as much happy if not less than any well fed and well equiped city person. This has helped to be more grateful to god and taught to count my blessings rather than cribbing for the things that I don’t have. I hope that the efforts I have put in through Melghat Mitra have brought some positive changes in life of Korkus. I think the project has worked because we were never an NGO seeking to bring about drastic reforms. We went as friends and we remain so.

If one loves animals, then seeing an ad on television depicting animal cruelty can tug at the heart and make the anger boil. However, these ads are on television for one purpose; to make people more aware of the plight of animals and perhaps to convince more people to become active in organizations that are against animal cruelty. Seeing one of the similar ads on the TV stirred me to the core and I thought to join an organization for animal rights to channel my anger positively.
The BSPCA (Bombay Society for the Prevention of Cruelty to Animals) in Mumbai is a charitable organization in existence since 1874. Its purpose is to prevent cruelty to animals and provide help and relief to all animals in Mumbai city. The animal hospital works 24 hours a day and treats an average of about 400 animals per day which includes different species of animals.

The BSPCA is effectively controlling cruelties to animals in Mumbai by employing SPCA Inspectors, social workers and by way of spreading education and awareness programmes. Volunteers are essential part of the hospital team for successfully running these programmes. As a Volunteer since 2 years I got opportunities such as to be a Foster mom or dad for litters of puppies ,Dog walking by-weekdays and weekend ,Grooming for dogs and horses, Fundraising ,Dog kennel assistant-cleaning kennels, feeling water bowls ,to be Emergency help and rescue correspondent ,Data entry operator, to help and participate in the events like Marathon etc. Most cruelty perpetrated on animals is needless and can be avoided with just a little thoughtfulness and sensitivity. There has to be awareness, accompanied by kindness and concern towards all God's creatures. I am glad that I can do my bit for the animals that share our world through BSPCA.

Mahatma Gandhi spearheaded a lot of efforts for social reconstruction and worked for the betterment of disadvantaged. How do you see your education at London Met helping you to follow this leadership?

Around two decades after the first clinical evidence of Acquired Immunodeficiency Syndrome was reported, AIDS has become the most devastating disease that human kind has ever faced. Since the epidemic began, more than 30 million people have been infected with Human Immunodeficiency Virus (HIV). AIDS threatens every fabric of society, increasingly targeting the poor and underprivileged. The pandemic is out of control, we have no effective antiviral agent and no immediate prospect of a vaccine. India being the world's fourth largest population suffering from AIDS, I can feel the extreme need to do research in HIV. Currently investigators are conducting research on various areas of HIV especially its infection, pathogenesis, development and testing of preventive HIV vaccines, prevention strategies, and new therapeutic treatments for HIV infection and AIDS-associated opportunistic infections. This research is identifying new and more effective targets for drugs and vaccines. The desire to become a part of this war against HIV has motivated me to opt for a career in biomedical research in the field of HIV and its therapeutics.

I feel that the knowledge I have gained is infinitesimal and fragmentary. I realize that I am still languishing on the shores and yet to venture into the vast realm of Biomedical sciences. So I chose to go for Masters in the subject so that I can improve my understanding and knowledge related to HIV and AIDS. It’ll not only give me insight to wider perspectives but also guide me in achieving my goal.

The Gandhian method of non-violence is relevant more than ever before in contemporary global scenario. Give us one or two examples of how you have embodied these attitudes in your life.

On Nov 26 last year, 10 Pakistani terrorists sneaked into this mega polis Mumbai by the Arabian Sea, landing by rubber dinghy and unleashed mayhem for three days in this city. Their killing spree and the bloody saga ended only when security forces killed all but one of them - Mohammed Ajmal Amir Kasab was captured during the attack. The 60 hours of terror left 166 people dead. I lost my dear friend in the same. The pain was excruciating and used to surge after seeing her parents losing their only child. Of cores my first reaction was that of sheer anger and helplessness towards the callous terrorist. But soon I realised it was useless. Instead I participated in a candle-light vigil, a police parade, an all-religion prayer meet at the Gateway of India and a memorial service at Chabad House where people from all walks of life came together to pay tribute to the victims and heroes of the 26/11 terror attacks. These peace march were ofcores in memory of those who lost their lives in the mayhem but also a strong indication to terrorist that we were down but not out. As an alert and active citizen I take it up on my selves and tell others to do their bit to maintain vigilance and help the authorities to make the city safer by reporting unclaimed items, suspicious strangers or activities in the vicinity.

I participated in Annual National Service Camps organized by National Service Scheme (N.S.S) in S.I.E.S. College in 2004 and 2005. The Camps were set in villages in Maharashtra which were suffering from alcoholism. During our stay in the village we discovered that the government officials itself are culprits of increasing alcoholism in most of these areas. But I and my groups didn’t confront them directly. We made skits regarding awareness of bad effects of alcoholism and invited those officials along with villagers for the same. Thus I think it was a peaceful and eventually more effective way of tackling that situation.

NF-κB : A Future Drug Target

NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) is a transcription factor that started its life as a B cell-specific nuclear protein, but has matured to become one of the most extensively studied transcriptional regulators, found to control many pathophysiological processes. Its role ranges from its transcriptional regulatory functions to its participation in diverse processes, including inflammation, immunity, mucosal homeostasis and cancer. NF-kB regulates the expression of over 200 genes that are involved in a variety of functions. It is basically a protein complex found in almost all animal cell types and is involved in cellular responses to stimuli such as stress, cytokines, free radicals, ultraviolet irradiation, oxidized LDL, and bacterial or viral antigens.

NF-κB plays a key role in regulating the immune response to infection. It regulates the expression of cytokines, inducible nitric oxide synthase (iNOS), cyclo-oxgenase 2 (COX-2), growth factors, inhibitors of apoptosis and effector enzymes in response to ligation of many receptors involved in immunity including T-cell receptors (TCRs), B-cell receptors (BCRs) and members of the Toll-like receptor/IL-1 receptor super family. It also takes part in the development and the activity of a number of tissues including the central nervous system. Moreover, pathological dysregulation of NF-κB is linked to inflammatory and autoimmune diseases as well as cancer. Activation of the NF-κB/Rel transcription family, by nuclear translocation of cytoplasmic complexes, plays a central role in inflammation through its ability to induce transcription of proinflammatory genes. This pathway is activated upon appropriate cellular stimulation, most often by signals related to pathogens or stress.

In the below link, we will discuss the the regulation of NF-κB activity by IκB proteins and IκB kinase (IKK), specificity of various NF-κB proteins, their role in inflammatory disease, and the development of therapeutic strategies aimed at inhibition of NF-κB.

NF-κB A Future Drug Target.pdf

Cocaine

Introduction

Cocaine is chief alkaloid found in the leaves of a South American shrub, Coca. In olden days, coca leaves were used for their stimulant properties (chewed as it produced a numbing effect on mouth and tongue) by natives of south America, particularly those living at high altitude ,who used it to reduce fatigue during work at these altitudes .Considerable mystical significance was attached to the powers of cocaine to boost the flagging human spirit.

Chemist Freud tested cocaine extensively on his patients and his family. Observing the result of Freud’s experiments with cocaine, his ophthalmologist colleague, Koller, obtained supplies of the drug and discovered its local anaesthetic action, later its psycho stimulant effects was proved. These discoveries led to cocaine becoming at one time, the most frequently abused substances in Western countries. Though its use has decreased somewhat since 1990. In the International context, UNODC (United Nations Office on Drugs and Crime) report some 13.4 million people (0.3%) of the world’s population aged between 15 and 64 years have used cocaine in the year 2009.

The first clinical use of cocaine as the local anaesthetic agent occurred in 1884, where cocaine was employed as a topical agent for eye surgery as well as being used to produce a nerve block. The idea was rapidly taken up within a few years and cocaine as anaesthesia was introduced in dentistry and general surgery. In Britain, The Rolleston Act of 1926 gives doctor the right to prescribe narcotics on their own judgement, it become primary basis of narcotic legislation until 1967. A synthetic substitute, procaine a potent anaesthetic was discovered in 1905. Many other useful compounds were later developed and effective Narcotic Laws were passed.
Generally, local anaesthetic molecule like cocaine consists of an aromatic parent linked by an ester bond to the basic side chain. It is a weak base, with pKa values mainly in the range 8-9. Consequently, it is mainly but not completely, ionised at physiological pH. This is an important in relation to its ability to penetrate the nerve sheath and axon membrane. The presence of the ester bond in cocaine is important because of its susceptibility to metabolic hydrolysis. The ester-containing compounds are usually inactivated in the plasma and tissue (mainly liver) by non –specific esterases. The metabolism of Cocaine leads to productions of mainly Benzoylecgonine along with Ecgonine and Ecgonine methyl ester.

Pharmacodynamics:

The primary action of the cocaine is blocking of Na+ channel conductance.In the nerve that is actively conducting impulses, it inhibits the inward migration of Na+ Ions. Its blocks the initiation and propagation of action potentials by preventing the voltage dependent increase in Na+ conductance. Its main action is to block sodium channels, which they do by physically plugging the transmembrane pore, interacting with the residues of the S6 trans membrane helical domain. In general, conduction in small- diameter nerve fibres is blocked more readily than in large fibres. Since noninceptive impulses are carried by A delta-and C-fibres (small- diameter nerve fibres), pain sensation is blocked more readily than other sensory modalities (touch, proprioception, etc.). Motor axons, being large in diameter, are relatively resistant. Thus in the nerve that is actively conducting impulses, it inhibits the inward migration of Na+ Ions. This results in elevation of the threshold for electrical excitation, reduction in the rate of rise of the action potential, slowing of the propagation of the impulse. If the drug concentration is sufficiently high, there is complete block of conduction. Thus cocaine interferes with the process fundamental to the generation of the action potential, namely, the large transient, voltage dependent rise in the permeability of the membrane to Na+ ions.

Neuronal reuptake of released noradrenaline is blocked by Cocaine .This enhance the effects of both sympathetic nerve activity and circulating noradrenaline. Cocaine enhances sympathetic transmission, causing increased arterial pressure, excitement etc. It strongly potentiates the action of noradrenaline in experimental animals or in isolated tissues provided the sympathetic nerve terminals are intact. Thus cocaine acts as a local anaesthetic.

Negative Body effects:

Cocaine enhances the peripheral effects of sympathetic nerve activity producing euphoria, garrulousness, increased motor activity and a magnification of pleasure. With excessive dosage, tremors and convulsions followed by respiratory and vasomotor depression may occur. They produce a mixture of stimulant and depressant effects on the CNS, often resulting in restlessness and tremor, with subjective effects ranging from confusion to extreme agitation The peripheral sympathomemitic actions leads to tachycardia, vasoconstriction and increase in the blood pressure . The main acute dangers are cardiac dysrhythmias and coronary or cerebral thrombosis. Slowly developing damage to the myocardium can also occur, leading to heart failure, even in absence of acute cardiac effects. Cocaine produces no clear cut physical dependence syndrome but tends to depression and dysphoria coupled with craving for the drug following the initial stimulant effect. Withdrawal of cocaine after administration for a few days causes a marked deterioration of motor performance and learned behaviour, which is restored by resuming dosage of the drug. There is therefore a considerable degree of psychological dependence. The pattern of dependence evolving from the occasional use through escalating dosage to compulsive binges. The duration of action of cocaine about 30 minutes when given intravenously. Other unwanted effects that are specific to particular drug include mucosal irritation in case of cocaine.

Cocaine can severely impair brain development in utero. The brain size is significantly reduced in babies exposed to cocaine in pregnancy, and the incidence of neurological and limb malformations also increased. The incidence of ischemic and haemorrhagic brain lesions, and of sudden infant death, is also higher in cocaine exposed babies.Cocaine has rapid onset of action (1min) and duration up to 2 hrs and Plasma half life of 1 hr, depending on the dose and route of administration used.

Pharmacokinetics

• Administration:
  
  Cocaine is mainly available in two forms, cocaine powder (hydrochloride salt) and crack (freebase). It is readily absorbed by many routes. Cocaine powder (Cocaine-HCl) is cut into ‘lines’ (ca 25mg) and is usually administered by snorting through the nose (IN) via straw, spoon ,rolled up banknote etc. Cocaine may also be made into a solution and injected either on its own or in combination with heroin (known as a ‘Speedball’). Cocaine use increased dramatically when the free base form “crack” is produced by ‘washing’ the salt with ammonia or mixing it with sodium bicarbonate and is smoked in a pipe. As per the report, there were three main methods of consuming cocaine in 2003 and these were: snorting or sniffing (70%), injecting (17%), and smoking (11%).
  

• Absorption and Distribution:
  
  The rate of absorption of a drug into the bloodstream is affected by the dose administered, the vascularity at the site of injection, and the specific physiochemical properties of the drug itself.Bioavailability or Peak plasma concentration of cocaine for different route of administration is different.
  
  For e.g. Route of Administration--- Bioavailability--- Peak Plasma Concentration
  

Injection (IV) ---------------100%------------ 0.20-0.40mg/L(65mg dose)
Snorting (IN)--------------- 25-95%---------- 0.20-0.40mg/L(100mg dose)
Smoking (SM)-------------- 57-70%---------- 0.20-0.40mg/L (100mg dose)
Orally (PO) ---------------- 20%------------- 0.10-0.50mg/L (100mg dose)

Table 1: Bioavailability or Peak plasma concentration of cocaine for different route of administration. Plasma concentration of 5.0mg/L observed in fatalities.

All tissues will be exposed to cocaine after absorption, but the concentration achieved will vary among the different organs. Although the highest concentrations appear to occur in the more highly perfused organs (i.e. brain, kidney and lung), factors such as degree of protein binding and lipid solubility also affect drug distribution. The lung can absorb as much as 90 percent of cocaine during the first pass. Consequently, it acts as a buffer to prevent higher and, therefore, more toxic concentrations that would occur otherwise. Ester drug, cocaine is almost completely metabolized by liver before excretion. However, the total dose administered and the degree of drug accumulation resulting from the initial and subsequent doses are still of concern.

Placental transfer of local anaesthetics is known to occur rapidly, the foetal blood concentrations generally reflects those found in the mother. However, the quantity of drug crossing to the foetus is also related to the time of exposure, that is, from the time of injection to delivery.

• Decomposition and Clearance:
  
  It is mainly by means of pseudocholinesterase of the blood and partly by liver enzymes resulting into a number of metabolites like benzoylecgonine, ecgonidine, norecgonidine methyl ester and m-hydroxyl-benzoylecgonine. Among these benzoylecgonine is the major metabolite, which is formed both non enzymatically as well as through the action of esterase found in a number of tissues including hepatocytes, the enzymatic mechanism being the dominant one, after which the metabolite is excreted via the kidney. Hence urine is majorly used as sample for detection.
  
  A cocaine metabolite is also deposited in hair, and analysis of its content along the hair shaft allows the pattern of cocaine consumption to be monitored, a technique which has revealed a much higher incidence of cocaine use than was voluntarily reported. Cocaine metabolites are also found in sweat of the abuse.
  

Drug Analysis:

• Need of Analysis:
  
  If there is a ‘new’ patient we need to: Determine the subjects current drug use. If an established patient we need to: First, check compliancy with prescription or abstinence. Second, determine any ‘extra-curricular’ drug use and then check for any ‘Sample adulteration’ aimed at minimising positive analytical results. If a patient presenting in a hospital A&E, the drug analysis is done to: First treat the patient’s symptoms.Then to obtain biological specimens to allow determination if any Drugs of Abuse are present. For Forensic purposes such as Screen analysis of drug is done to identify if any abused drugs are present and to determine concentrations in blood/tissues to potentially identify a cause of death.
  

a) Sample used for testing:

The studies carried out by Pascal Kintz in Strasbourg, France have shown that cocaine metabolites were present in greater concentrations than the parent drug for urine, and that in sweat and hair the parent drug was usually present in greater amounts. The duration of analyte is much higher in sweat and hair compared to urine. The appropriate and available sample should be used for the analysis of the drug.

b) Spot Test:

There are a number of presumptive tests for cocaine available from the literature. One of the Spot Test for Cocaine is done by using 5% (0.125M) NaOH in methanol. It reacts with cocaine to release methyl benzoate-detectable by its odour (oil of wintergreen). It is available in test kit form. Most widely used test is Cobalt Isothiocyanate test. It is simple test where there is the addition of the material under investigation to a 2% (wt/vol) solution of cobalt isothiocyanate in water. The presence of cocaine and related alkaloids results in blue colour being formed. The scott test is the modification of the cobalt isothiocyanate test and involves a 2% (wt/vol) solution of cobalt isothiocyanate in water, dilutes in equal volume of glycerine, concentrated HCl and chlororform. In order to the test the sample for the presence of cocaine, a small amount of material is placed in a test- tube and 5 drops of the first reagent are added. A blue colour develops if cocaine is present. One drop of concentrated HCl is then added and the blue colour, if it results from the presence of cocaine, should disappear, leaving a pink solution. Several drops of chloroform should then be added. An intense blue colour forms in the chloroform (lower) layer if cocaine is present. Negative and positive controls should be performed concurrently with analysis of the test sample. The Scott test gives greater discrimination and specificity towards cocaine.

• Method of analysis: Methods of Analysis

a) Immunoassay:

Immunoassays are tests that use antibodies to identify and measure analytes. In forensic science typically a biological sample is screened for the presence of an antigen such as a drug. Immunoassays are commonly uses for screening purposes because they are readily amenable to large batch analysis, are relatively sensitive, and require little or no sample preparation. Because immunoassays are targeted to detect benzoylecgonine, they are particularly well suited for screening urine specimens. There are several types of immunoassays on the market, depending on the product selected, for e.g. Fluorescence polarization immunoassay (FPIA), Enzyme immunoassay (EIA), Micropartical immunoassay (KIMS), Radioimmunoassay (RIA), Cloned Enzyme Donor Immunoassay (CEDIA), Enzyme Linked Immunosorbant Assay (ELISA). In addition to their use for urine analysis, some of these assays have been successfully adapted to blood tissue analysis. Although, all immunoassay techniques are targeted on benzoylecgonine, cross reactivities varies considerably by manufacturers and analytical principle, thus Immunoassay is chosen according to need. For e.g. Immunoassays that possess substantial cross reactivity to cocaine and ethylcocaine, such as the Daignostic Products corporation (DPC, Los Angeles) RIA assay, are particularly useful for screening blood. The detection limit for cocaine and ethyl cocaine using the DPC RIA is as low as 0.010mg/L depending on lot. In addition the assay can readily detect benzoylecgonine at 0.100 mg /L. The FPIA assay show considerable cross reactivity to m –hydroxybenzoylecgonine, making it well suited to screening meconium. Although the cut off concentration for benzoylecgonine in federal workplace drug test is 0.300 mg/L, most immunoassays can detect lower concentration reliably. Cocaines most common cross reactive substance are ecgonine and ecgonine methyl ester.

b) Chromatographical Methods:

1. Sample preparation:

For the cocaine and its metabolites to be analyzed using chromatographic techniques , the drugs must be separated form the biological matrix. Hence, the sample preparation is required. This may be accomplished either by liquid-liquid or solid-phase extraction procedures. The latter techniques can be readily adapted to laboratory automation devices.
There are several important issues that must be considered before selecting an extraction procedure. Cocaine is ester that is susceptible to hydrolysis in alkaline conditions and at elevated temperatures. In addition, plasma and liver esterases also contribute to hydrolysis. During sample preparation, it is critical that the amount of time the biological specimen remains in conditions that are unfavourable to cocaine stability be minimized; otherwise, esters may hydrolize in vitro and complicate the interpretation of analytical results. Consideration must also be given to the targeted analytes because there is considerable range in polarity for cocaine and its metabolites.

Other than stability concerns, the extraction of cocaine and ethylcocaine is straightforward. These compounds can be readily extracted into n- butyl chloride at the pH of 8-9 by Liquid/ liquid phase extraction. Here chloroform isopropanol mixture (9:1) is used. These conditions will also extract bezoylecgonine and ecgonine methyl ester, although not at optimal recoveries. Solid phase extraction (SPE) procedures, also used for these analytes commonly use a protein precipitation step before a buffered supernatant is applied to the extraction column. Elution is typically accomplished with mixture of methylene chloride, isopropanol and ammonium hydroxide (78:20:2). Ecgonine requires SPE containing anion exchange media. Their elution solvents have also been used, depending on the analytes targeted and the column used.

2. Methods:

- Thin Layer Chromatography (TLC):

Thin layer chromatography (TLC) is a simple technique that can be used to analyze for both cocaine and benzoylecgonine. It is generally limited to analysis of urine specimens due to lack of sensitivity. In fact, TLC has higher detection limits than immunoassay. After extracting 5 ml of urine for basic drugs and spotting the concentrated extract on a silica TLC plate, one can expect a sensitivity of about 1-2 mg/ml (3.4-6.9 micromol/L) for benzoylecgonine using the standard Davidow solvent with visualisation by either Dragendorff’s reagent and/or iodoplatinate. Sensitivity for the cocaine by TLC is generally better than for benzoylecgonine using most basic drug extraction schemes. High performance TLC results in considerably improved detection limits. As a qualitative tool, TLC may be used to confirm immunoassay results when positive forensic identification is not required.TLC is particularly useful for benzoylecgonine because it does not migrate up the plate very far, allowing it to be readily distinguished from many other basic drugs.In TLC of cocaine , the Liquid/Liquid extraction of urine/bile is done .The Spot concentration extract on silica TLC plate used is 1-2mg/L.It is developed with 1.5%NH3/methanol and visualized with UV254 or acidified potassium iodoplatinate.

- Gas Chromatography:

Gas chromatography is a separation technique most frequently used for the analysis of cocaine and its metabolites .Cocaine can be readily assayed without derivatisation using nitrogen- phosphorus detection and detection by Mass spectrometry (MS) in both the electron impact and positive chemical ionization modes when coupled with Gas Chromatography. Flame ionization detection may also be used, but is not nearly as sensitive. Chromatography may be improved by derivatisation of cocaine to the p-nitrococaine or p-fluorococaine derivative using p- nitrobenzoylchloride or p-flourorbenzoylchloride reply, in modification of the Schotten- Baumann reaction.

Electron capture detectors are very sensitive and have been used for analysis of benzoylecgonine after acylation.The major disadvantage to this detector is that cocaine must be reduced before acylation. Detection by MS provides the highest specificity of all GC detectors and is nearly requirement for the forensic confirmation of cocaine and metabolites today.

- Liquid Chromatography:

Liquid chromatography is very useful because cocaine, ethylcocaine and benzoylecgonine can be analysed simultaneously without derivatisation when using UV detection. UV has a sensitivity of 0.05 mg/L(0.17 micromol/L) for cocaine. But there is low selectivity and it does not allow the use of deuterated internal standards.

On the other hand ,LC coupled with MS may be the ideal separation and detection techniques for cocaine and its metabolites without need for derivatisation.LC/MS is still not commonly used due to cost and maintenance issues.

- Mass spectrometry:

Mass spectrometry (MS) is an analytical technique that measures the mass-to-charge ratio of charged particles. Thus it is used for determining masses of particles, for determining the elemental composition or chemical structures of a sample or molecule and for detection and Quantification of drug in the sample.

Generally, MS instruments consist of three modules: An ion source, which can convert gas phase sample molecules into ions by fragmentation. A mass analyzer, which sorts the ions by their masses by applying electromagnetic fields. A detector, which measures the value of an indicator quantity, provides data for calculating the abundances of each ion present. Thus the technique has both qualitative and quantitative uses.

- Cocaine Fragmentation:

MS is now in very common use in analytical laboratories that study physical, chemical, or biological properties of a great variety of compounds. To determine the presence of cocaine and/or these metabolites, urine samples are screened using enzyme immunoassay, and positive results are confirmed using gas chromatography/mass spectrometry. Mass spectrometry is widely used for confirmation because MS data provide positive identification based on mass spectral information, and can be used as confirming evidence in courts of law. A particular advantage with MS detection is that deuterated analogs of cocaine e.g Norcocaine and its major metabolite are now readily available and can be used as internal standards by virtue of having chemical characteristics nearly identical to their non deutereated analogs. They allow for brilliant reproducibility and accurate analysis. Thus MS is coupled with GC or LC give excellent results.
In GC/MS ,cocaine and ethylcocaine assayed without derivatization while Benzoylecgonine/ecgonine require derivatization (Trimethylsilyl).The detection is carried out using Chemical ionization or Electron impact detection. The assay used helium as carrier gas, a 30-m bonded phase fused silica OV-1 capillary column, and solid injection at 290°C evaporator temperature. Sensitivity obtained was <0.01mg/ml when sample was human hair. Although GC/MS methods are well established, and provide excellent confirmation data, analysis time is long and sample preparation, which includes derivatization, is labour-intensive and time-consuming. However LC-MS benzoylecgonine and ecgonine can be directly assayed, thus save time and efforts. The limits of detection for LC with Tandem MS ranged from 3 to 23 ng /ml and the limits of quantitation ranged from 7 to 69 ng/ml.

A lot research is going on in improving the detection resulting into more sensitive and accurate method. One of such successful endeavour is Matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI.). This method was used to image the distribution of cocaine and its metabolites in intact single hair samples from chronic users down to a concentration of 5 ng/mg. A less commonly used, but equally valuable, chromatographic method is high performance liquid chromatography coupled with mass spectrometry (HPLC/MS). The analytes do not have to be derivatized for this approach, making HPLC/MS more attractive to the analyst. A well-chosen stationary phase and a mobile phase with a high organic content allow efficient desolvation and ionization in the MS, and permit detection of cocaine and its metabolites at low picogram levels on-column. Under optimal screening conditions, limits of detection of 1ng/ml for cocaine and 5ng/ml for benzoylecgonine have been reported to be 10pg and 50pg on-column, respectively (10µL injection) for the C18 stationary phase. HPLC/MS, like GC/MS, provides reproducible, reliable data that can be used in court. The HPLC/MS method developed in this investigation provides symmetric peaks and excellent sensitivity in a reduced analysis time.

In Drug analysis these are devices that include a biological component and a transducer. The transducer converts the detection made by the biological component into an electrical signal that is a function of the concentration of the analyte. Often this achieved by immobilising the biological component on to the transducer. Because these biological components are specific to an analyte this gives specificity to the biosensor and means that they can used on samples that have not been pre-treated (to remove interfering compounds).Enzymes were the first catalysts used in biosensors. In Bioreceptors, the binding is non-catalytic and irreversible. The development of these biosensors took off with the increasing availability of monoclonal antibodies. One of the papers discussing this new technique was regarding Analyte 2000, a four-channel fibre optic biosensor (FOB) for analysis of cocaine and its metabolites (COC) in human urine using a competitive fluorescence immunoassay where fluorescence is produced by evanescent excitation of bound cyanine dye-tagged antimouse antibody (CY5-Ab). The effective concentration range of benzoylecgonine (BE) for inhibiting the fluorescent signals was 0.75-50 ng/ml, with IC50 of 9.0 ng/ml.

Use of Drug Analysis:

Both bulk samples and trace materials may be encountered by the forensic scientist. For the bulk samples, the analytical sequence includes Physical description, presumptive tests, TLC and confirmation using GC/MS. Interestingly, although the presumptive testing stage may include colour tests, another method can also be employed where the odour is noted following the addition of sodium hydroxide in methanol.

Under Sports law, cocaine is classified under stimulants leading to performance enhancement. Most drugs are banned outright in Olympic competitions. The World Anti-Doping Agency oversees world-wide doping and has a list of prohibited substances. Sample testing for first time carried out in1972 Munich Olympic Games was carried out with GC with nitrogen-selective detection. Then in following years GC/MS was used to confirm the presence of anabolic steroids. In ordered to be accredited a laboratory must be able to carry out: GC, GC-MS, HPLC, TLC, Immunoassays.

Cocaine has negative effects on body especially CNS, heart etc leading to driving fatalities due lack of concentration, coordination etc. The field tests are designed accordingly. Field Impairment Tests currently in use checks for physical signs: Pupil Size should be between 3 and 6.5 mm. Romberg Test- Involves standing with feet together, Test checking balance and co-ordination. Further confirmation is done by Testing Urine for the Presence of abused drugs. Primary screening which is usually by immunoassay. Cocaine is confirmed by TLC.

An individual who ingests wrapped packets of illicit drugs to transport them by internal concealment is called Body Pack. Generally, the analyses is carried out by CEDIA immunoassay screening of Urine and colon faeces for Cocaine and confirmed by GC/MS.Blood was screened for Basic drugs, stimulant drugs, acidic drugs, benzodiazepines and morphine.