Monoclonal antibody therapy

biochemist INDIA (31 Oct 011): The main objective is stimulating the patient's immune system to attack the malignant tumor cells and the prevention of tumor growth by blocking specific cell receptors.

Variations exist within this treatment, e.g.radioimmunotherapy a radioactive dose directly to the target cell, and lethal chemical doses to the target. Structure and function of human and therapeutic antibodies Immunoglobulin G (IgG) antibodies are large heterodimeric molecules, approximately 150 kDa and are composed of two different kinds of polypeptide chain, called the heavy (~50kDa) and the light chain (~25kDa).

There are two types of light chains, kappa (κ) and lambda (λ).

By cleavage with enzyme papain, the Fab (fragment-antigen binding) part can be separated from the Fc (fragment crystalline) part of the molecule.

The Fab fragments contain the variable domains, which consist of three hypervariable amino acid domains responsible for the antibody specificity embedded into constant regions.

There are four known IgG subclasses all of which are involved in Antibody-dependent cellular cytotoxicity. The immune system responds to the environmental factors it encounters on the basis of discrimination between self and non-self.

Tumor cells are not specifically targeted by one's immune system since tumor cells are the patient's own cells.

Tumor cells, however are highly abnormal, and many display unusual antigens that are either inappropriate for the cell type, its environment, or are only normally present during the organisms' development (e.g.

fetal antigens). Other tumor cells display cell surface receptors that are rare or absent on the surfaces of healthy cells, and which are responsible for activating cellular signal transduction pathways that cause the unregulated growth and division of the tumor cell.

Examples include ErbB2, a constitutively active cell surface receptor that is produced at abnormally high levels on the surface of approximately 30% of breast cancer tumor cells.

Such breast cancer is known a HER2 positive breast cancer. Antibodies are a key component of the adaptive immune response, playing a central role in both in the recognition of foreign antigens and the stimulation of an immune response to them.

The advent of monoclonal antibody technology has made it possible to raise antibodies against specific antigens presented on the surfaces of tumors.

Therapy For Alzheimer Disease: CpG DNA

biochemist INDIA (31 oct 011):

Alzheimer disease is the most common form of dementia, affecting approximately 1.6% of the population in the United States (nearly 19% in the 75-84 age group). It is an incurable, degenerate, and terminal disease thought to be caused by accumulation of oligomeric amyloid β (oAβ).

Microglia are the resident immune cells in the central nervous system; they remove damaged neurons, plaques, and infectious agents from the brain and spinal cord. Microglia cluster around senile Aβplaques in Alzheimer disease patients; however, the role of microglia in oAβtoxicity remains unclear. Doi et al discovered that microglial activation with unmethylated CpG DNA, which binds to an immune receptor on microglia, prevented oAβtoxicity and enhanced oAβ peptide clearance in culture. Furthermore, injection of CpG DNA directly into the brain mitigated both cognitive impairment and learning defects in a mouse model of Alzheimer disease. CpG DNA may therefore be a therapeutic candidate for treatment of Alzheimer disease.

Researchers conclude that "CpG, especially class B and C, may also be effective therapeutic agents against oAβ1-42 neurotoxicity in [Alzheimer disease]."

Alzheimer's Disease Risk and Amyloid Beta Toxicity

BIOCHEMIST INDIA (30 OCT 011): In a development that sheds new light on the pathology of Alzheimer's disease (AD), a team of Whitehead Institute scientists has identified connections between genetic risk factors for the disease and the effects of a peptide toxic to nerve cells in the brains of AD patients.

The scientists, working in and in collaboration with the lab of Whitehead Member Susan Lindquist, established these previously unknown links in an unexpected way. They used a very simple cell type -- yeast cells -- to investigate the harmful effects of amyloid beta (Aβ), a peptide whose accumulation in amyloid plaques is a hallmark of AD. This new yeast model of Aβ toxicity, which they further validated in the worm C. elegans and in rat neurons, enables researchers to identify and test potential genetic modifiers of this toxicity.

"As we tackle other diseases and extend our lifetimes, Alzheimer's and related diseases will be the most devastating personal challenge for our families and one the most crushing burdens on our economy," says Lindquist, who is also a professor of biology at Massachusetts Institute of Technology and an investigator of the Howard Hughes Medical Institute. "We have to try new approaches and find out-of the-box solutions."

In a multi-step process, reported in the journal Science, the researchers were able to introduce the form of Aβ most closely associated with AD into yeast in a manner that mimics its presence in human cells. The resulting toxicity in yeast reflects aspects of the mechanism by which this protein damages neurons. This became clear when a screen of the yeast genome for genes that affect Aβ toxicity identified a dozen genes that have clear human homologs, including several that have previously been linked to AD risk by genome-wide association studies (GWAS) but with no known mechanistic connection.

With these genetic candidates in hand, the team set out to answer two key questions: Would the genes identified in yeast actually affect Aβ toxicity in neurons? And if so, how?

To address the first issue, in a collaboration with Guy Caldwell's lab at the University of Alabama, researchers created lines of C. elegans worms expressing the toxic form of Aβ specifically in a subset of neurons particularly vulnerable in AD. This resulted in an age-dependent loss of these neurons. Introducing the genes identified in the yeast that suppressed Aβ toxicity into the worms counteracted this toxicity. One of these modifiers is the homolog of PICALM, one of the most highly validated human AD risk factors. To address whetherPICALM could also suppress Aβ toxicity in mammalian neurons, the group exposed cultured rat neurons to toxic Aβ species. Expressing PICALM in these neurons increased their survival.

The question of how these AD risk genes were actually impacting Aβ toxicity in neurons remained. The researchers had noted that many of the genes were associated with a key cellular protein-trafficking process known as endocytosis. This is the pathway that nerve cells use to move around the vital signaling molecules with which they connect circuits in the brain. They theorized that perhaps Aβ was doing its damage by disrupting this process. Returning to yeast, they discovered that, in fact, the trafficking of signaling molecules in yeast was adversely affected by Aβ. Here again, introducing genes identified as suppressors of Aβ toxicity helped restore proper functioning.

Much remains to be learned, but the work provides a new and promising avenue to explore the mechanisms of genes identified in studies of disease susceptibility.

"We now have the sequencing power to detect all these important disease risk alleles, but that doesn't tell us what they're actually doing, how they lead to disease," says Sebastian Treusch, a former graduate student in the Lindquist lab and now a postdoctoral research associate at Princeton University.

Jessica Goodman, a postdoctoral fellow in the Lindquist lab, says the yeast model provides a link between genetic data and efforts to understand AD from the biochemical and neurological perspectives.

"Our yeast model bridges the gap between these two fields," Goodman adds. "It enables us to figure out the mechanisms of these risk factors which were previously unknown."

Members of the Lindquist lab intend to fully exploit the yeast model, using it to identify novel AD risk genes, perhaps in a first step to determining if identified genes have mutations in AD patient samples. The work will undoubtedly take the lab into uncharted territory.

Notes staff scientist Kent Matlack: "We know that Aβ is toxic, and so far, the majority of efforts in the area of Aβ have been focused on ways to prevent it from forming in the first place. But we need to look at everything, including ways to reduce or prevent its toxicity. That's the focus of the model. Any genes that we find that we can connect to humans will go into an area of research that has been less explored so far."

This work was supported by an HHMI Collaborative Innovation Award, an NRSA fellowship, the Cure Alzheimer's Fund, the National Institutes of Health, the Kempe foundation, and Alzheimerfonden

Duchenne muscular dystrophy (DMD): role of gene therapist.

Duchenne muscular dystrophy (DMD) is associated with mutations in the dystrophin gene that disrupt the open reading frame whereas the milder Becker's form is associated with mutations which leave an in-frame mRNA transcript that can be translated into a protein that includes the N- and C- terminal functional domains.

It has been shown that by excluding specific exons at, or adjacent to, frame-shifting mutations, open reading frame can be restored to an out-of-frame mRNA, leading to the production of a partially functional Becker-like dystrophin protein. Such targeted exclusion can be achieved by administration of oligonucleotides that are complementary to sequences that are crucial to normal splicing of the exon into the transcript.

This principle has been validated in mouse and canine models of DMD with a number of variants of oligonucleotide analogue chemistries and by transduction with adeno-associated virus (AAV)-small nuclear RNA (snRNA) reagents encoding the antisense sequence. Two different oligonucleotide agents are now being investigated in human trials for splicing out of exon 51 with some early indications of success at the biochemical level.

http://www.nature.com/mt/journal/v19/n1/abs/mt2010219a.html

Biochemist India: Methylenetetrahydrofolate reductase gene polymorph...

Biochemist India: Methylenetetrahydrofolate reductase gene polymorph...: Cerebral palsy (CP) covers a group of non-progressive chronic disorders of motor function and posture caused by lesions of the developing fe...

Methylenetetrahydrofolate reductase gene polymorphisms and cerebral palsy in Chinese infants

Cerebral palsy (CP) covers a group of non-progressive chronic

disorders of motor function and posture caused by lesions of the

developing fetal or infant brain. CP is the most common cause of severe physical disability in childhood, occurring in 1–2/1000 live births. many cases are multifactorial in origin and exhibit marked etiologic heterogeneity. Risk factors for CP can be categorized as prenatally, perinatally and postnatally acquired of which about 70– 80% are acquired prenatally. CP may be related to genomic factors, as well as to environmental incursions during brain development. Methylenetetrahydrofolate reductase (MTHFR) catalyses irreversibly the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. Studies showed that MTHFR gene polymorphisms are associated with inherited thrombophilias, which can result in adverse pregnancy outcomes such as CP.

Researchers observed a significant difference in allele and genotype frequencies between CP + MR patients and controls at rs4846049, rs1476413 and rs1801131 and there was a statistically significant difference in the frequencies of the three SNPs between CP + MR and CP-only cases.

this is the first report to our knowledge to demonstrate that MTHFR genetic polymorphisms are associated

with CP combined with MR. It adds to the existing evidence that certain gene variants may in some way contribute to CP

UGA scientists team up to define first-ever sequence of biologically important carbohydrate

If genes provide the blueprint for life and proteins are the machines that do much of the work for cells, then carbohydrates that are linked to proteins are among the tools that enable cells to communicate with the outside world and each other.

But until now, scientists have been unable to determine the structure of a biologically important so-called GAG proteoglycan – or even to agree whether these remarkably complex molecules have well-defined structures.

In a paper published in the early online edition of Nature Chemical Biology, however, a team of scientists from the University of Georgia and Rensselaer Polytechnic Institute announced that it has, for the first time, determined the sequence and structure of a glycosaminoglycan, or GAG, proteoglycan.

“The fact that a structure even exists is surprising, because people had the sense that the complexity of these molecules pointed to a randomness,” said study co-author Jonathan Amster, professor and head of the department of chemistry in the UGA Franklin College of Arts and Sciences. “There are many different areas in medicine that will be enabled by understanding carbohydrates at this fundamental level.”

Modifications to the GAG, or carbohydrate biopolymer, portion of proteoglycans have been associated with the presence and malignancy of certain cancers, for example, and the researchers noted that the identification of carbohydrates that are involved in disease opens the door to the development of drugs that can block their action.

The field of glycobiology is still in its infancy, largely because attempts to sequence proteoglycans have, until now, ended in frustration and failure. A small sample of DNA can be amplified many times, and its sequence, or arrangement of molecules, can be determined quickly with modern tools. DNA is simply a set of instructions for making proteins, so a sample of DNA also can allow scientists to produce copious quantities of protein for study.

Carbohydrates, however, are a bit messier. Scientists don’t fully understand how cells create them, and a given proteoglycan exists in multiple forms that are similar but not quite the same.

The researchers chose the simplest known GAG proteoglycan, a compound known as bikunin that is used in Japan for the treatment of acute pancreatitis, for their study. Of course, simplicity is a relative term: the sugar is composed of up to 55 distinct carbohydrate rings, which means that there are 210 billion different sequence possibilities. Previous studies performed over the past five years by the researchers that identified common sequences within the carbohydrate decreased the expected number of sequences to a mere 43 million.

Past attempts to sequence proteoglycans have relied on the so-called ‘bottom up’ approach in which scientists use enzymes to chop a molecule into its component parts and then try to put it back together, like a puzzle. Using an alternative approach known as the ‘top down’ method, the scientists placed the compound into high-powered mass spectrometers in both the Amster and Linhardt labs that allowed them to break the compound in predictable places. With larger puzzle pieces to work with, the scientists were able to deduce the structure of bikunin.

“Now that we have demonstrated that bikunin, a small chondroitin sulfate proteoglycan, has sequence, we are moving on to larger, more structurally complex dermatan sulfate and heparan sulfate proteoglycans,” said study co-author Robert Linhardt, professor at Rensselaer Polytechnic Institute. “These show important biological activities in development and in cancer, and we are optimistic that our sequencing approach will work here as well.”

Like all groundbreaking scientific discoveries, the finding actually raises more questions than it answers. Amster explained that the addition of sulfate to the sugar, for example, could in principle occur anywhere along the carbohydrate chain. What the researchers found, however, was that the sites of sulfation occur only in particular rings. “That was the unexpected finding,” Amster said, “because based on the current understanding of biology, there is no known mechanism for controlling that type of specificity.”

As they work to determine the structure of more complex proteoglycans, the scientists hope that their findings will encourage other researchers to consider the role that they play in health.

“We know that carbohydrates are how cells communicate with each other and their environment, but they’re also likely to play many roles that we can’t even envision yet,” Amster said. “And in order to understand them, we need to be able to study them at this molecular level.”