Proteomics and Spinal Fluid

 

Scientists and doctors have a far better understanding of the proteins in healthy spinal fluid, thanks to a U.S., Swedish team who identified 2,630 proteins in the clear fluid that protects the brain and spinal cord. This discovery nearly triples the number of proteins known to exist in spinal fluid. Another striking finding was that slightly more than half of the proteins were not found in blood.

The team was led by Richard D. Smith, Ph.D., of Pacific Northwest National Laboratory, and Steven E. Schutzer, MD, of the University of Medicine and Dentistry of New Jersey—New Jersey Medical School.

In conducting this research, the team used integrated resources at EMSL that included a custom-built automated nanocapillary liquid chromatography system coupled on-line to one of two mass spectrometers, modified in-house with an electrodynamic ion funnel.

 

 

Proteome

 

The proteome is the entire set of proteins expressed by a genome, cell, tissue or organism. More specifically, it is the set of expressed proteins in a given type of cells or an organism at a given time under defined conditions. The term is a portmanteau of proteins and genome.

The term has been applied to several different types of biological systems. A cellular proteome is the collection of proteins found in a particular cell type under a particular set of environmental conditions such as exposure to hormone stimulation. It can also be useful to consider an organism’s complete proteome, which can be conceptualized as the complete set of proteins from all of the various cellular proteomes. This is very roughly the protein equivalent of the genome. The term “proteome” has also been used to refer to the collection of proteins in certain sub-cellular biological systems. For example, all of the proteins in a virus can be called a viral proteome.

The proteome is larger than the genome, especially in eukaryotes, in the sense that there are more proteins than genes. This is due to alternative splicing of genes and post-translational modifications like glycosylation or phosphorylation.

Moreover the proteome has at least two levels of complexity lacking in the genome. While the genome is defined by the sequence of nucleotides, the proteome cannot be limited to the sum of the sequences of the proteins present. Knowledge of the proteome requires knowledge of (1) the structure of the proteins in the proteome and (2) the functional interaction between the proteins.

Proteomics, the study of the proteome, has largely been practiced through the separation of proteins by two dimensional gel electrophoresis. In the first dimension, the proteins are separated by isoelectric focusing, which resolves proteins on the basis of charge. In the second dimension, proteins are separated by molecular weight using SDS-PAGE. The gel is dyed with Coomassie Brilliant Blue or silver to visualize the proteins. Spots on the gel are proteins that have migrated to specific locations.

The mass spectrometer has augmented proteomics. Peptide mass fingerprinting identifies a protein by cleaving it into short peptides and then deduces the protein’s identity by matching the observed peptide masses against a sequence database. Tandem mass spectrometry, on the other hand, can get sequence information from individual peptides by isolating them, colliding them with a non-reactive gas, and then cataloguing the fragment ions produced.

 

 

 

Proteomics – What is Proteomics?

 

PDB structure 2p69, one of the protein structures solved by the New York SGX Research Center for Structural Genomics, a large scale PSI center. This human phosphatase is involved in vitamin B6 metabolism .

 

 

 

Proteomics is the large-scale study of proteins, particularly their structures and functions. Proteins are vital parts of living organisms, as they are the main components of the physiological metabolic pathways of cells.

The term “proteomics” was first coined in 1997 to make an analogy with genomics, the study of the genes. The word “proteome” is a blend of “protein” and “genome”, and was coined by Marc Wilkins in 1994 while working on the concept as a PhD student.

The proteome is the entire complement of proteins, It is now known that mRNA is not always translated into protein, and the amount of protein produced for a given amount of mRNA depends on the gene it is transcribed from and on the current physiological state of the cell. Proteomics confirms the presence of the protein and provides a direct measure of the quantity present.

Scientists are very interested in proteomics because it gives a much better understanding of an organism than genomics. First, the level of transcription of a gene gives only a rough estimate of its level of expression into a protein.

An mRNA produced in abundance may be degraded rapidly or translated inefficiently, resulting in a small amount of protein. Second, as mentioned above many proteins experience post-translational modifications that profoundly affect their activities; for example some proteins are not active until they become phosphorylated.

Methods such as phosphoproteomics and glycoproteomics are used to study post-translational modifications. Third, many transcripts give rise to more than one protein, through alternative splicing or alternative post-translational modifications. Fourth, many proteins form complexes with other proteins or RNA molecules, and only function in the presence of these other molecules. Finally, protein degradation rate plays an important role in protein content.

Post-translational modifications

Not only does the translation from mRNA cause differences, many proteins are also subjected to a wide variety of chemical modifications after translation. A lot of these post-translational modifications are critical to the protein’s function.

Phosphorylation

One such modification is phosphorylation, which happens to many enzymes and structural proteins in the process of cell signaling. The addition of a phosphate to particular amino acids—most commonly serine and threonine mediated by serine/threonine kinases, or more rarely tyrosine mediated by tyrosine kinases—causes a protein to become a target for binding or interacting with a distinct set of other proteins that recognize the phosphorylated domain.

Because protein phosphorylation is one of the most-studied protein modifications many “proteomic” efforts are geared to determining the set of phosphorylated proteins in a particular cell or tissue-type under particular circumstances. This alerts the scientist to the signaling pathways that may be active in that instance.

Ubiquitination

Ubiquitin is a small protein that can be affixed to certain protein substrates by enzymes called E3 ubiquitin ligases. Determining which proteins are poly-ubiquitinated can be helpful in understanding how protein pathways are regulated. This is therefore an additional legitimate “proteomic” study. Similarly, once it is determined what substrates are ubiquitinated by each ligase, determining the set of ligases expressed in a particular cell type will be helpful.

Additional modifications

Listing all the protein modifications that might be studied in a “Proteomics” project would require a discussion of most of biochemistry; therefore, a short list will serve here to illustrate the complexity of the problem.

In addition to phosphorylation and ubiquitination, proteins can be subjected to (among others) methylation, acetylation, glycosylation, oxidation and nitrosylation. Some proteins undergo ALL of these modifications, often in time-dependent combinations, aptly illustrating the potential complexity one has to deal with when studying protein structure and function.

Distinct proteins are made under distinct settings

Even if one is studying a particular cell type, that cell may make different sets of proteins at different times, or under different conditions. Furthermore, as mentioned, any one protein can undergo a wide range of post-translational modifications.

Therefore a “proteomics” study can become quite complex very quickly, even if the object of the study is very restricted. In more ambitious settings, such as when a biomarker for a tumor is sought – when the proteomics scientist is obliged to study sera samples from multiple cancer patients – the amount of complexity that must be dealt with is as great as in any modern biological project.

 

Robotic preparation of MALDI mass spectrometry samples on a sample carrier.

 

 

Complexity of the problem
After genomics, proteomics is considered the next step in the study of biological systems. It is much more complicated than genomics mostly because while an organism’s genome is more or less constant, the proteome differs from cell to cell and from time to time. This is because distinct genes are expressed in distinct cell types. This means that even the basic set of proteins which are produced in a cell needs to be determined.

In the past this was done by mRNA analysis, but this was found not to correlate with protein content. It is now known that mRNA is not always translated into protein,and the amount of protein produced for a given amount of mRNA depends on the gene it is transcribed from and on the current physiological state of the cell. Proteomics confirms the presence of the protein and provides a direct measure of the quantity present.
Post-translational modifications
Not only does the translation from mRNA cause differences, many proteins are also subjected to a wide variety of chemical modifications after translation. Many of these post-translational modifications are critical to the protein’s function.

Phosphorylation

One such modification is phosphorylation, which happens to many enzymes and structural proteins in the process of cell signaling. The addition of a phosphate to particular amino acids—most commonly serine and threonine mediated by serine/threonine kinases, or more rarely tyrosine mediated by tyrosine kinases—causes a protein to become a target for binding or interacting with a distinct set of other proteins that recognize the phosphorylated domain.

Because protein phosphorylation is one of the most-studied protein modifications, many “proteomic” efforts are geared to determining the set of phosphorylated proteins in a particular cell or tissue-type under particular circumstances. This alerts the scientist to the signaling pathways that may be active in that instance.

Ubiquitination

Ubiquitin is a small protein that can be affixed to certain protein substrates by enzymes called E3 ubiquitin ligases. Determining which proteins are poly-ubiquitinated can be helpful in understanding how protein pathways are regulated. This is therefore an additional legitimate “proteomic” study. Similarly, once it is determined which substrates are ubiquitinated by each ligase, determining the set of ligases expressed in a particular cell type will be helpful.

Additional modifications

Listing all the protein modifications that might be studied in a “Proteomics” project would require a discussion of most of biochemistry; therefore, a short list will serve here to illustrate the complexity of the problem. In addition to phosphorylation and ubiquitination, proteins can be subjected to (among others) methylation, acetylation, glycosylation, oxidation and nitrosylation. Some proteins undergo ALL of these modifications, often in time-dependent combinations, aptly illustrating the potential complexity one has to deal with when studying protein structure and function.

Distinct proteins are made under distinct settings

Even if one is studying a particular cell type, that cell may make different sets of proteins at different times, or under different conditions. Furthermore, as mentioned, any one protein can undergo a wide range of post-translational modifications.

Therefore a “proteomics” study can become quite complex very quickly, even if the object of the study is very restricted. In more ambitious settings, such as when a biomarker for a tumor is sought – when the proteomics scientist is obliged to study sera samples from multiple cancer patients – the amount of complexity that must be dealt with is as great as in any modern biological project.

Limitations to genomic study

Scientists are very interested in proteomics because it gives a much better understanding of an organism than genomics. First, the level of transcription of a gene gives only a rough estimate of its level of expression into a protein. An mRNA produced in abundance may be degraded rapidly or translated inefficiently, resulting in a small amount of protein. Second, as mentioned above many proteins experience post-translational modifications that profoundly affect their activities; for example some proteins are not active until they become phosphorylated. Methods such as phosphoproteomics and glycoproteomics are used to study post-translational modifications. Third, many transcripts give rise to more than one protein, through alternative splicing or alternative post-translational modifications. Fourth, many proteins form complexes with other proteins or RNA molecules, and only function in the presence of these other molecules. Finally, protein degradation rate plays an important role in protein content.

 

Methods of studying proteins

 

Determining proteins which are post-translationally modified

One way in which a particular protein can be studied is to develop an antibody which is specific to that modification. For example, there are antibodies which only recognize certain proteins when they are tyrosine-phosphorylated, known as phospho-specific antibodies; also, there are antibodies specific to other modifications. These can be used to determine the set of proteins that have undergone the modification of interest.

For sugar modifications, such as glycosylation of proteins, certain lectins have been discovered which bind sugars. These too can be used.

A more common way to determine post-translational modification of interest is to subject a complex mixture of proteins to electrophoresis in “two-dimensions”, which simply means that the proteins are electrophoresed first in one direction, and then in another, which allows small differences in a protein to be visualized by separating a modified protein from its unmodified form. This methodology is known as “two-dimensional gel electrophoresis“.

Recently, another approach has been developed called PROTOMAP which combines SDS-PAGE with shotgun proteomics to enable detection of changes in gel-migration such as those caused by proteolysis or post translational modification.

Determining the existence of proteins in complex mixtures

Classically, antibodies to particular proteins or to their modified forms have been used in biochemistry and cell biology studies. These are among the most common tools used by practicing biologists today.

For more quantitative determinations of protein amounts, techniques such as ELISAs can be used.

For proteomic study, more recent techniques such as matrix-assisted laser desorption/ionization (MALDI) have been employed for rapid determination of proteins in particular mixtures and increasingly electrospray ionization (ESI).

Computational methods in studying protein biomarkers

Computational predictive models have shown that extensive and diverse feto-maternal protein trafficking occurs during pregnancy and can be readily detected non-invasively in maternal whole blood. This computational approach circumvented a major limitation, the abundance of maternal proteins interfering with the detection of fetal proteins, to fetal proteomic analysis of maternal blood. Computational models can use fetal gene transcripts previously identified in maternal whole blood to create a comprehensive proteomic network of the term neonate. Such work shows that the fetal proteins detected in pregnant woman’s blood originate from a diverse group of tissues and organs from the developing fetus. The proteomic networks contain many biomarkers that are proxies for development and illustrate the potential clinical application of this technology as a way to monitor normal and abnormal fetal development.

An information theoretic framework has also been introduced for biomarker discovery, integrating biofluid and tissue information. This new approach takes advantage of functional synergy between certain biofluids and tissues with the potential for clinically significant findings not possible if tissues and biofluids were considered individually. By conceptualizing tissue-biofluid as information channels, significant biofluid proxies can be identified and then used for guided development of clinical diagnostics. Candidate biomarkers are then predicted based on information transfer criteria across the tissue-biofluid channels. Significant biofluid-tissue relationships can be used to prioritize clinical validation of biomarkers.

Establishing protein–protein interactions

Most proteins function in collaboration with other proteins, and one goal of proteomics is to identify which proteins interact. This is especially useful in determining potential partners in cell signaling cascades.

Several methods are available to probe protein–protein interactions. The traditional method is yeast two-hybrid analysis. New methods include protein microarrays, immunoaffinity chromatography followed by mass spectrometry, dual polarisation interferometry, Microscale Thermophoresis and experimental methods such as phage display and computational methods

 

Practical applications of proteomics

 

An example of a protein structure determined by the Argonne Midwest Center for Structural Genomics  —  deposits 1,000th protein structure

 

 

 

One of the most promising developments to come from the study of human genes and proteins has been the identification of potential new drugs for the treatment of disease. This relies on genome and proteome information to identify proteins associated with a disease, which computer software can then use as targets for new drugs. For example, if a certain protein is implicated in a disease, its 3D structure provides the information to design drugs to interfere with the action of the protein. A molecule that fits the active site of an enzyme, but cannot be released by the enzyme, will inactivate the enzyme. This is the basis of new drug-discovery tools, which aim to find new drugs to inactivate proteins involved in disease. As genetic differences among individuals are found, researchers expect to use these techniques to develop personalized drugs that are more effective for the individual.

Biomarkers

The FDA defines a biomarker as, “A characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention”.

Understanding the proteome, the structure and function of each protein and the complexities of protein–protein interactions will be critical for developing the most effective diagnostic techniques and disease treatments in the future.

An interesting use of proteomics is using specific protein biomarkers to diagnose disease. A number of techniques allow to test for proteins produced during a particular disease, which helps to diagnose the disease quickly. Techniques include western blot, immunohistochemical staining, enzyme linked immunosorbent assay (ELISA) or mass spectrometry.

Proteogenomics

In what is now commonly referred to as proteogenomics, proteomic technologies such as mass spectrometry are used for improving gene annotations. Parallel analysis of the genome and the proteome facilitates discovery of post-translational modifications and proteolytic events, especially when comparing multiple species (comparative proteogenomics).

Current research methodologies

Fluorescence two-dimensional differential gel electrophoresis (2-D DIGE)can be used to quantify variation in the 2-D DIGE process and establish statistically valid thresholds for assigning quantitative changes between samples.

Comparative proteomic analysis can reveal the role of proteins in complex biological systems, including reproduction. For example, treatment with the insecticide triazophos causes an increase in the content of brown planthopper (Nilaparvata lugens (Stål)) male accessory gland proteins (Acps) that can be transferred to females via mating, causing an increase in fecundity (i.e. birth rate) of females.To identify changes in the types of accessory gland proteins (Acps) and reproductive proteins that mated female planthoppers received from male planthoppers, researchers conducted a comparative proteomic analysis of mated N. lugens females. The results indicated that these proteins participate in the reproductive process of N. lugens adult females and males.

Proteome analysis of Arabidopsis peroxisomes has been established as the major unbiased approach for identifying new peroxisomal proteins on a large scale.

There are many approaches to characterizing the human proteome, which is estimated to contain between 20,000 and 25,000 non-redundant proteins. The number of unique protein species likely increase by between 50,000 and 500,000 due to RNA splicing and proteolysis events, and when post-translational modification are also considered, the total number of unique human proteins is estimated to range in the low millions.

In addition, first promising attempts to decipher the proteome of animal tumors have recently been reported.

U.S. Department of Health and Human Services
NATIONAL INSTITUTES OF HEALTH NIH News
National Institute of Mental Health (NIMH)

 

 

 

NEURONS GROWN FROM SKIN CELLS MAY HOLD CLUES TO AUTISM

 

 

Representative iPSC-derived neurons from Timothy syndrome patient (bottom) shows increased numbers of neurons that produce the chemical messengers norepinephrine and dopamine, compared to those from a control subject (top).(Medical Daily Ricardo Dolmetsch, Ph.D., Stanford University)

 

 

 

Rare syndrome’s workings could help explain how brain wiring goes awry — NIH-funded study

 

 

Potential clues to how autism miswires the brain are emerging from a study of a rare, purely genetic form of the disorders that affects fewer than 20 people worldwide.  Using cutting-edge “disease-in a-dish”(http://www.nimh.nih.gov/about/director/2011/skin-cells-to-neurons-disease-in-a-dish-promises-shortcut-to-discovery.shtml) technology, researchers funded by the National Institutes of Health have grown patients’ skin cells into neurons to discover what goes wrong in the brain in Timothy Syndrome (http://ghr.nlm.nih.gov/condition/timothy-syndrome). Affected children often show symptoms of autism spectrum disorders (http://www.nimh.nih.gov/health/topics/autism-spectrum-disorders-pervasive-developmental-disorders/index.shtml) along with a constellation of physical problems.

 

Abnormalities included changes in the composition of cells in the cortex, the largest brain structure in humans, and of neurons that secrete two key chemical messengers.  Neurons that make long-distance connections between the brain’s hemispheres tended to be in short supply.

 

Most patients with Timothy Syndrome meet diagnostic criteria for an autism spectrum disorder. Yet, unlike most cases of autism, Timothy syndrome is known to be caused by a single genetic mutation.

 

“Studying the consequences of a single mutation, compared to multiple genes with small effects, vastly simplifies the task of pinpointing causal mechanisms,” explained Ricardo Dolmetsch, Ph.D. (http://www.nimh.nih.gov/media/video/dolmetsch.shtml), of Stanford University, a National Institute of Mental Health (NIMH) grantee (http://projectreporter.nih.gov/project_info_description.cfm?aid=8206064&icde=1032931) who led the study. His work was partially funded by a NIH Director’s Pioneer Award (http://projectreporter.nih.gov/project_info_description.cfm?aid=8136230&icde=10319997).

 

Dolmetsch, and colleagues, report on their findings Nov. 27, 2011 in the journal Nature Medicine.

 

“Unlike animal research, the cutting-edge technology employed in this study makes it possible to pinpoint molecular defects in a patient’s own brain cells,” said NIMH Director Thomas R. Insel, M.D. “It also offers a way to screen more rapidly for medications that act on the disordered process.”

 

Prior to the current study, researchers knew that Timothy syndrome is caused by a tiny glitch in the gene that codes for a calcium channel protein in cell membranes. The mutation results in too much calcium entering cells, causing a tell-tale set of abnormalities throughout the body.  Proper functioning of the calcium channel is known to be particularly critical for proper heart rhythm — many patients die in childhood of arrhythmias — but its role in brain cells was less well understood.

 

To learn more, Dolmetsch and colleagues used a new technology called induced pluripotent stem cells (iPSCs) (http://stemcells.nih.gov/info/basics/basics10.asp). They first converted skin cells from Timothy Syndrome patients into stem cells and then coaxed these to differentiate into neurons.

 

“Remarkable reproducibility” observed across multiple iPSC lines and individuals confirmed that the technique can reveal defects in neuronal differentiation — such as whether cells assume the correct identity as the brain gets wired-up in early development, said the researchers.  Compared to those from controls, fewer neurons from Timothy Syndrome patients became neurons of the lower layers of the cortex and more became upper layer neurons.  The lower layer cells that remained were more likely to be the kind that project to areas below the cortex. In contrast, there were fewer-than-normal neurons equipped to form a structure, called the corpus callosum, which makes possible communications between the left and right hemispheres.

 

Many of these defects were also seen in parallel studies of mice with the same genetic mutation found in Timothy syndrome patients. This supports the link between the mutation and the developmental abnormalities.

 

Several genes previously implicated in autism were among hundreds found to be expressed abnormally in Timothy Syndrome neurons. Excess cellular calcium levels also caused an overproduction of neurons that make key chemical messengers. Timothy Syndrome neurons secreted 3.5 times more norepinephrine and 2.3 times more dopamine than control neurons. Addition of a drug that blocks the calcium channel reversed the abnormalities in cultured neurons, reducing the proportion of catecholamine-secreting cells by 68 percent.

 

The findings in Timothy Syndrome patient iPSCs follow those in Rett Syndrome, another single gene disorder that often includes autism-like symptoms. About a year ago, Alysson Muotri, Ph.D., and colleagues at University of California, San Diego, reported deficits (http://www.eurekalert.org/pub_releases/2010-11/uoc–urc_1110410.php) in the protrusions of neurons, called spines, that help form connections, or synapses. The Dolmetsch team’s discovery of earlier (neuronal fate) and later (altered connectivity) defects suggest that disorders on the autism spectrum affect multiple stages in early brain development.

 

“Most of these abnormalities are consistent with other emerging evidence that ASDs arise from defects in connectivity between cortex areas and show decreased size of the corpus callosum,” said Dolmetsch.  “Our study reveals how these might be traceable to specific mechanisms set in motion by poor regulation of cellular calcium. It also demonstrates that neurons derived from iPSCs can be used to identify the cellular basis of a neurodevelopmental disorder.”

 

The mechanisms identified in this study may become potential targets for developing new therapies for Timothy Syndrome and may also provide insights into the neural basis of deficits in other forms of autism, said Dolmetsch.

 

The mission of the NIMH is to transform the understanding and treatment of mental illnesses through basic and clinical research, paving the way for prevention, recovery and cure. For more information, visit the NIMH website <http://www.nimh.nih.gov/index.shtml>.

 

About the National Institutes of Health (NIH): NIH, the nation’s medical research agency, includes 27 Institutes and Centers and is a component of the U.S. Department of Health and Human Services. NIH is the primary federal agency conducting and supporting basic, clinical, and translational medical research, and is investigating the causes, treatments, and cures for both common and rare diseases. For more information about NIH and its programs, visit <www.nih.gov>.

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REFERENCE:

Using iPS cell-derived neurons to uncover cellular phenotypes associated with Timothy Syndrome. Pasca SP, Portmann T, Voineagu I, Yazawa M, Shcheglovitov O, Pasca AM, Cord B, Palmer TD, Chikahisa S, Seiji N, Bernstein JA, Hallmayer J, Geschwind DH, Dolmetsch RE. November 27, 2011. Nature Medicine.

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The html version of this release contains images of:

–a catecholamine-producing neurons at <http://www.nimh.nih.gov/images/news-items/dolmetsch-timothy-iPSC-catechol.jpg>

— a corpus collosum at: <http://www.nimh.nih.gov/images/news-items/dolmetsch-timothy-iPS-collosum.jpg>

Target Health launched Target e*CRF in 1999, and last week, the FDA approved the 8th drug  which Target e*CRF was used for key aspects of the development process. In addition to the 8 drug approvals, there have been 2 PMA device and 10 PMA diagnostic approvals. And the best is yet to come.

 

This past year, Target Health launched 1) Target e*Studio™, which is the technology transfer version of Target e*CRF, and 2) Target e*CTR™, which allows for the seamless integration, in clinical trials, of direct data entry, eSource and the EDC database. Target Health expects to submit the first NDA ever, in Q1 2013, which used eSource in lieu of paper patient records.

 

Other software products include Target Document® (eTMF), Target Encoder® (MedDRA/WhoDrug coding); Target e*Pharmacovigilance™ (3500A and CIOMS forms), Target e*CTMS™, Target Monitoring Reports™, etc.

 

For more information about Target Health contact Warren Pearlson (212-681-2100 ext. 104). For additional information about software tools for paperless clinical trials, please also feel free to contact Dr. Jules T. Mitchel or Ms. Joyce Hays. The Target Health software tools are designed to partner with both CROs and Sponsors. Please visit the Target Health Website at www.targethealth.com

Eli Hurvitz, Founder of Teva, Dies at 79

 

 

Eli Hurvitz, who began his career washing lab equipment at a drug company and went on to build Teva Pharmaceutical Industries into the largest generic drug maker in the world, has died. While no cause was given, in an interview with the newspaper Yediot Aharonot earlier this year Mr. Hurvitz said he had cancer. “I am not the type to surrender,” he told the paper. “That is how it was in business, and that is how it is with the illness.”

 

Mr. Hurvitz started out in the 1950s at Assia, a small pharmaceutical company partly owned by his father-in-law, in Petah Tikva, east of Tel Aviv. He became managing director in 1976 and merged Assia with two other small firms, Zori and Teva, to form the company now known as Teva, which means “nature” in Hebrew. Mr. Hurvitz was the president and chief executive of Teva for the next 25 years. Through aggressive mergers and acquisitions, a reputation for quality and low prices, Teva grew into a giant that supplied one of every six prescriptions dispensed in the United States. It has a presence in 60 countries and 45,000 employees. In 2010, it reached $16 billion in sales.

 

Eliyahu Hurvitz was born in Jerusalem in 1932 and moved to Tel Aviv with his family as a child. When the state of Israel was founded and the Arab-Israeli war broke out in 1948, Mr. Hurvitz was drafted and spent time on a kibbutz as part of a unit that established agricultural settlements. He then went on to study economics at a branch of the Hebrew University of Jerusalem in Tel Aviv. In 1953, he married Dalia Solomon and began working at Assia.

 

Mr. Hurvitz was awarded the Israel Prize in 2002 for lifetime achievement and his contributions to the state and society. In addition to his business, he had been chairman of Bank Leumi, president of the Manufacturers Association of Israel and chairman of the Israel Democracy Institute, an independent research institute in Jerusalem.

Chewing Gum Cuts Ear Infection Risk in Kids

 

 

 

Ear infections are extremely common, especially in runny-nosed kids. The latest research indicates that when young children get 1) ___, they end up with an ear infection 61% of the time. Chewing gum containing xylitol may actually prevent ear infections in kids. Xylitol is a sugar alcohol derived from the sugar xylose. Xylitol comes from birch bark. An alternative sweetener, Xylitol helps prevent cavities and sweetens foods made for diabetics. It contains 40% fewer calories than sugar and still provides a sweet flavor.

 

Xylitol originates from the fibers in fruits and vegetables, including mushrooms, raspberries, strawberries, yellow plums, lettuce and cauliflower. In fruits and vegetables, xylitol constitutes less than 1%, according to a study led by researcher R. Sreenivas Rao. The act of chewing and 2) ___ assists with the disposal of earwax and clearing the middle ear, while the presence of xylitol prevents the growth of bacteria in the eustachian tubes (auditory or pharyngotympanic tubes) which connect the 3) ___ and ear.

 

When bacteria enter the body, they adhere to the tissues using a variety of sugar complexes. The open nature of xylitol and its ability to form many different sugar-like structures appears to interfere with the ability of many bacteria to adhere. In a double-blind, randomized, controlled trial, saline solutions of xylitol significantly reduced the number of nasal coagulase-negative Staphylococcus bacteria. The study attributed the benefits to the increased effectiveness of endogenous (naturally present in the body) antimicrobial factors. In a small clinical trial nasally administered xylitol reduced ear complaints in children previously having chronic complaints, on the order of almost one a month, by more than 92%. Beneficial effects on asthma with nasal administration, have also been reported.

 

In a meta-analysis of three Finnish studies, children who chewed 4) ___ — or took other products laden with xylitol, including lozenges or syrup — had about a 25% lower risk of developing acute otitis media compared with control interventions, Amir Azarpazhooh, DMD, of the University of Toronto, and colleagues reported in Cochrane Reviews. “Based on the studies we reviewed, xylitol seems to be a promising alternative to conventional therapies to prevent acute otitis media among healthy 5) ___,” they wrote.

 

Acute otitis media is the most common infection for which kids are treated with 6) ___, which has spurred concerns over antibiotic resistance. So researchers have searched for alternative means of prevention or treatment, not all of which have been successful. Xylitol, or birch sugar, has been one such alternative. It’s a five-carbon polyol sugar alcohol found in a number of fruits, which has been shown to inhibit the growth and acid production of certain bacteria, particularly S. mutans. It is for this feature that some dentists recommend it for preventing 7) ___, the researchers said.

 

Since a key step in the pathogenesis of otitis media is the colonization of the upper airway with 8) ___ that move from the nasopharynx to the middle ear via the eustachian tubes, the researchers hypothesized that it may be effective for preventing middle ear infections. So they conducted a review and meta-analysis of four studies: three randomized controlled trials in 1,826 Finnish children, and another among 1,277 Finnish children in day care who had a respiratory infection. In a meta-analysis of the three trials, the researchers saw a reduced risk of acute otitis media in children who were given 8 to 10 g/day of xylitol in any form — either as gum, a lozenge, or syrup — compared with control interventions (RR 0.75). In the fourth trial, however, xylitol had no effect on reducing the occurrence of acute otitis media in kids with upper respiratory infection.

 

Gum appeared to work best; in healthy children, chewing xylitol was superior to syrup at preventing otitis media (RR 0.59), though it had no advantages if given during respiratory infection. There were no differences between xylitol lozenges and syrups in preventing ear infections in healthy kids or in those who had respiratory infections, and there was no difference between gum and lozenges for preventing 9) ___ infections in healthy kids or in those with a respiratory infection. The authors noted that the study was limited because the data arise from a small number of studies, mainly from the same research group.

 

A number of factors prevent xylitol chewing gum from being used more widely to prevent ear infection. First, school rules against chewing gum may hamper its preventive use in a place where it may be needed most. Also, previous surveys have shown that only about half of clinicians know about the medical uses of xylitol.

Still, Azarpazhooh and colleagues concluded that a daily dose of about 8 g of xylitol — potentially as two pieces of chewing gum five times a day after meals for at least five minutes — can prevent acute otitis media in kids without acute upper respiratory 10) ___.

 

ANSWERS: 1) colds; 2) swallowing; 3) nose; 4) gum; 5) children; 6) antibiotics; 7) cavities; 8) bacteria; 9) ear; 10) infection

Richmond’s Medical Miracle

 

Photo Source: Library of Congress Chimborazo Hospital, Richmond, Va.

 

 

During the opening months of the Civil War, the streets of Richmond, Va., filled with bloodied bodies. The thousands of Confederate wounded were treated in a range of makeshift hospitals hastily established in hotels, factories and private homes. But by autumn, as hopes the conflict would be brief faded, it became clear a war of this magnitude required a modernized medical response. That fall Samuel P. Moore, the Confederate surgeon general, secured both the facilities and the personnel to provide such a response at Chimborazo, a 40-acre plateau just east of the Confederate capital’s stately Church Hill neighborhood. The site got its name from Mount Chimborazo, an inactive volcano in Ecuador, famous at the time after being “discovered” by the German explorer-scientist Alexander von Humboldt.

 

Occupying 150 buildings, it was one of the largest hospitals in the world, typically serving around 4,000 sick and wounded soldiers at a time. Over the next three and a half years it treated 77,000 patients — twice the entire population of Richmond at the outbreak of the war. But Chimborazo was remarkable for more than just its gargantuan size. It would prove to be among the world’s most efficient, modern and sanitary hospitals of the period, an achievement due in no small part to Moore’s appointment of James B. McCaw to run the facility. The son, grandson and great-grandson of physicians, McCaw embodied the emergence of the modern, professional doctor. After attending medical school in New York, he returned to his native Richmond, accepting a professorship at the Medical College of Virginia and editorship of the Virginia Medical and Surgical Journal. His nascent understanding of the hygienic importance of cleanliness, his ties to M.C.V., the only medical school in the Confederacy to remain operational throughout the war, allowing Chimborazo to pioneer the practices of a teaching hospital, and his talent as an administrator proved invaluable in superintending Chimborazo.

 

McCaw organized Chimborazo into five divisions, each with its own surgeon-in-chief. Within each were 90 wards measuring about 80 feet by 28 feet and containing 40 patient beds. The wards were spaced along 40-foot-wide avenues and 10-foot alleys, with 3 doors and 10 windows on the long sides of the buildings to provide ventilation. During the most propinquitous and bloody campaigns, when the number of wounded exceeded the capacity of the wards, 100 Sibley tents were pitched nearby, accommodating up to 10 patients each. Once the tents filled, additional patients were bivouacked in the open air.

 

McCaw Library. An 1863 map of Chimborazo Hospital.

 

The complex also included bathhouses, ice houses, carpentry and blacksmith shops, a soap manufactory, a stable, a chapel, an apothecary shop, a bakery that produced 10,000 loaves daily, a brewery that produced 400 kegs of beer at a time, and five dead houses, one for each of the divisions. Chimborazo maintained a large vegetable garden on a nearby farm, as well as herds of goats and cows. McCaw even secured a canal boat to travel the James River, bringing provisions from as far away as Lexington, Va.

 

In addition to the dozens of physicians, who were organized into a hierarchy of assistant surgeons, surgeons and surgeons-in-charge, the hospital employed enormous nursing and support staffs. Military hospitals had previously been manned largely by convalescing soldiers, but the labor needs at Chimborazo were so great — as was the pressure for any able soldiers to return to the front — that McCaw relied heavily on slaves, free blacks and white women to keep the hospital running.

 

Indeed, African-American labor was paramount to the running of Confederate military hospitals. Though the Civil War is often thought of as the watershed that opened the field of nursing to American women, female nursing was largely a Union, and not a Confederate, phenomenon; 9,000 women served as nurses in Union hospitals, compared to only 1,000 in Confederate hospitals, primarily because the use of hired-out male slaves in the South preempted the recruitment of white women.

 

McCaw repeatedly advertised for slave labor throughout the war, and he pleaded with Moore for impressment of Chimborazo’s enslaved staff, to prevent owners from removing the slaves in his service. Slaves and a smaller number of free blacks cooked, cleaned, and worked in manufacturing – tasks similar to those they’d long been assigned in non-hospital settings. But enslaved men also served in the nursing staff, an arrangement that shocked some of the patients.

 

White women did play a crucial role at Chimborazo, serving as matrons on the wards. Matrons oversaw the preparation and distribution of medicine and meals, as ordered by the physicians. They also provided a range of patient care, from reading Bible passages to writing letters home, and fielding requests for everything from a haircut to a “b’iled sweet pur-r-rta-a-a-tu-ur,” as Phoebe Yates Pember, who served as matron from late 1862 through the fall of Richmond, recalled in her entertaining, if occasionally self-aggrandizing, 1879 memoir, “A Southern Woman’s Story: Life in Confederate Richmond.”

 

Unfortunately, the historical record hasn’t yielded anything analogous to McCaw’s official correspondence or Pember’s memoir to provide the perspective of the enslaved and free black staff regarding their wartime experience at Chimborazo. Nevertheless, in a significant post-bellum coda to the story of Chimborazo, the facility that was constructed with slave labor in October 1861 and employed hired-out slaves throughout the war was converted after the war into a freedmen’s school. Opened in June 1865, it served hundreds of African-American students, many of whom lived on site. Like other freedmen’s schools, Chimborazo attracted a broad swath of the free black population in the years following emancipation; the November 1869 register listed students between ages 4 and 29. Published in the NYTimes, November 23, 2011, by Lois Leveen

Dosing of Clopidogrel (Plavix) Based on CYP2C19 Genotype and the Effect on Platelet Reactivity in Patients with Stable Cardiovascular Disease

 

 

Clopidogrel (Plavix) is an oral, thienopyridine class antiplatelet agent used to inhibit blood clots in coronary artery disease (CAD), peripheral vascular disease, and cerebrovascular disease. The drug works by irreversibly inhibiting a receptor called P2Y12, an adenosine diphosphate ADP chemoreceptor. Adverse effects include hemorrhage, severe neutropenia, and thrombotic thrombocytopenic purpura (TTP).

 

It has been reported that variants in the CYP2C19 gene influence the pharmacologic and clinical response to the standard 75-mg daily maintenance dose. As a result, a study published in the Journal of the American Medical Association (2011;306:2221-2228) was performed. to test whether higher doses (up to 300 mg daily) improve the response to clopidogrel in the setting of loss-of-function CYP2C19 genotypes.

 

The study, ELEVATE-TIMI 56, was a multicenter, randomized, double-blind trial that enrolled and genotyped 333 patients with cardiovascular disease across 32 sites from October 2010 until September 2011. Study participants received maintenance doses of clopidogrel for 4 treatment periods, each lasting approximately 14 days, based on genotype. In total, 247 noncarriers of a CYP2C19*2 loss-of-function allele were to receive 75 and 150 mg daily of clopidogrel (2 periods each), whereas 86 carriers (80 heterozygotes, 6 homozygotes) were to receive 75, 150, 225, and 300 mg daily. The main outcome measures were platelet function test results (vasodilator-stimulated phosphoprotein [VASP] phosphorylation and VerifyNow P2Y12 assays) and adverse events.

 

Results showed that:

 

1. Treatment with 75 mg daily, CYP2C19*2 heterozygotes had significantly higher on-treatment platelet reactivity than did noncarriers (VASP platelet reactivity index [PRI]: mean, 70.0% vs. 57.5%, and VerifyNow P2Y12 reaction units [PRU]: mean, 225.6 vs. 163.6; P < .001 for both comparisons).

 

2. Among CYP2C19*2 heterozygotes, doses up to 300 mg daily significantly reduced platelet reactivity, with VASP PRI decreasing to 48.9% and PRU to 127.5 (P < .001 for trend across doses for both).

 

3. Whereas 52% of CYP2C19*2 heterozygotes were nonresponders (≥230 PRU) with 75 mg of clopidogrel, only 10% were nonresponders with 225 or 300 mg (P < .001 for both).

 

4. Clopidogrel, 225 mg daily, reduced platelet reactivity in CYP2C19*2 heterozygotes to levels achieved with standard clopidogrel, 75 mg, in noncarriers

 

5. In CYP2C19*2 homozygotes, even with 300 mg daily of clopidogrel, the mean VASP PRI was 68.3% and mean PRU, 287.0.

 

According to the authors, among patients with stable cardiovascular disease, tripling the maintenance dose of clopidogrel to 225 mg daily in CYP2C19*2 heterozygotes achieved levels of platelet reactivity similar to that seen with the standard 75-mg dose in noncarriers; in contrast, for CYP2C19*2 homozygotes, doses as high as 300 mg daily did not result in comparable degrees of platelet inhibition.

Effects on 11-Year Mortality and Morbidity of Lowering LDL Cholesterol with Simvastatin (Zocor, Merck)

 

 

Findings of large randomized trials have shown that lowering LDL cholesterol with statins reduces vascular morbidity and mortality rapidly. However, only limited evidence exists about the long-term efficacy and safety of statin treatment. As a result, an article published in The Lancet, Early Online Publication (23 November 2011), was performed to assess long-term efficacy and safety of lowering LDL cholesterol with statins. The study used data from the extended follow-up of the Heart Protection Study (HPS), to report cause-specific mortality and major morbidity in the in-trial and post-trial periods.

 

For the trial, 20,536 patients at high risk of vascular and non-vascular outcomes were allocated either 40 mg simvastatin daily or placebo, using minimized randomization. Mean in-trial follow-up was 5.3 years (SD 1.2), and post-trial follow-up of surviving patients yielded a mean total duration of 11 years (SD 0.6). The primary outcome of the long-term follow-up was first post-randomization major vascular event. Analysis was by intention to treat.

 

Results showed that allocation to simvastatin yielded an average reduction in LDL cholesterol of 1.0 mmol/L and a proportional decrease in major vascular events of 23% (p<0.0001), with significant divergence each year after the first. During the post-trial period (when statin use and lipid concentrations were similar in both groups), no further significant reductions were noted in either major vascular events. However, during the combined in-trial and post-trial periods, no significant differences were recorded in cancer incidence at all sites (0.98 or any particular site, or in mortality attributed to cancer (1.01) or to non-vascular causes (0.96).

 

According to the authors, more prolonged LDL-lowering statin treatment produces larger absolute reductions in vascular events. Moreover, even after study treatment, benefits persisted for at least 5 years without any evidence of emerging hazards. The authors emphasized that these findings provide further support for the prompt initiation and long-term continuation of statin treatment.

Reciprocal Seasonal Variation in Vitamin D Status and Tuberculosis Notifications in Cape Town, South Africa

 

 

Vitamin D deficiency is associated with susceptibility to tuberculosis (TB) in HIV-uninfected people in Europe, but it is not known whether such an association exists among HIV-infected people in subtropical Africa. As a result, a study, published in the Proceedings of the National Academy of Sciences (2011;108:19013-19017), was conducted to determine whether vitamin D deficiency was associated with susceptibility to active TB in HIV-uninfected (n = 196) and HIV-infected (n = 174) black Africans in Cape Town, South Africa. The study also investigated whether there was evidence of seasonal variation in vitamin D status and TB notifications in this setting over an 8-year period.

 

Results showed that vitamin D deficiency (serum 25-hydroxyvitamin D [25(OH)D] <50 nmol/L) was present in 232 (62.7%) of 370 participants and was associated with active TB in both HIV-uninfected (odds ratio = 5.2; P < 0.001) and HIV-infected (odds ratio = 5.6; P < 0.001) people.

 

Vitamin D status also varied according to season: The mean serum 25(OH)D concentration was highest in January through March and lowest in July through September (56.8 vs. 30.7 nmol/L, respectively; P < 0.001). Reciprocal seasonal variation in TB notifications was observed: The mean number of TB notifications per quarter for Cape Town in 2003 to 2010 was lowest in April through June and highest in October through December (4,222 vs. 5,080; P < 0.001).

 

According to the authors, Vitamin D deficiency is highly prevalent among black Africans in Cape Town and is associated with susceptibility to active TB both in the presence and absence of HIV infection. The authors added that reciprocal seasonal variation in serum 25(OH)D concentration and TB notifications suggests that seasonal variations in vitamin D status and TB incidence in this setting may be causally related.

FDA approves Eylea for Wet Age-Related Macular Degeneration (AMD)

 

TARGET HEALTH excels in Regulatory Affairs and Public Policy issues. Each week we highlight new information in these challenging areas.

 

Congratulations to our friends and colleagues at Regeneron!

 

Wet (neovascular) age-related macular degeneration AMD gradually destroys a person’s sharp, central vision and is a leading cause of vision loss and blindness in Americans ages 60 and older. AMD affects the macula, the part of the eye that allows people to see fine detail needed to do daily tasks such as reading and driving. There are two forms of AMD, a wet form and a dry form. The wet form of AMD includes the growth of abnormal blood vessels. The blood vessels can leak fluid into the central part of the retina, also known as the macula. When fluid leaks into the macula, the macula thickens and vision loss occurs. An early symptom of wet AMD occurs when straight lines appear to be wavy.

 

The FDA has approved Eylea (aflibercept) to treat patients with AMD. The safety and effectiveness of Eylea was evaluated in two clinical trials involving 2,412 adult patients. People in the study received either Eylea or Lucentis (ranibizumab injection). The primary endpoint in each study was a patient’s clearness of vision (visual acuity) after one year of treatment.

 

Eylea is injected into the eye either every four weeks or every eight weeks by an ophthalmologist. The studies showed that Eylea was as effective as Lucentis in maintaining or improving visual acuity.

 

The most commonly reported side effects in patients receiving Eylea included eye pain, blood at the injection site (conjunctival hemorrhage), the appearance of floating spots in a person’s vision (vitreous floaters), clouding of the eye lens (cataract), and an increase in eye pressure. Eylea should not be used in those who have an active eye infection or active ocular inflammation. Eylea has not been studied in pregnant women, so the treatment should be used only in pregnant women if the potential benefits of the treatment outweigh any potential risks. Age related macular degeneration does not occur in children and Eylea has not been studied in children.

 

Other FDA-approved treatment options for wet AMD include: Visudyne (verteporfin for injection) approved in 2000, Macugen (pegaptanib sodium injection) approved in 2004, and Lucentis (ranibizumab injection) approved in 2006.

 

For more information: NEI: Facts about Age-Related Macular Degeneration

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