Posts Tagged ‘Medicine’

Muscular dystrophies are a class of degenerative diseases characterized by progressive muscular weakening, myopathy and paralysis.  Among these, one of the most famous is Duchenne muscular dystrophy (DMD), which affects about 1 in every 3000 males born in the United States.  This class of diseases faces the very limits of medical therapy, as there are currently no clinical treatments available to halt the progression of symptoms.  With only supportive care available, patients are left to progressively worsen, typically ending with loss of physical mobility, the ability to breathe independently and usually fatal heart related complications.  The disease maligns quality and length of life, such that most only live into their twenties and are confined to a wheelchair around their teens.  Only in very rare circumstances have any lived into their 30’s or 40’s.

It wasn’t until fairly recently that an accurate understanding of the pathology involved became clearer.  With DMD, mutations from reading frame shifts in the gene producing the dystrophin protein were found to be the root of the problem.  Dystrophin is a critical protein involved in muscle structural support and intracellular signaling with the extra-cellular matrix.  For any kind of contraction, a muscle must have an anchor and this protein helps play this part by linking the strand to a support structure called the DAP complex, which in turn links to the extracellular matrix.  Like a car without shock absorbers, muscle cells are more susceptible to physical membrane damage and slowly whither under the stress.  Damaged, leaky membranes are a classic indicator of failed cell viability and seen as the hallmark of dying cells, a fact corroborated by typical diagnostic staining procedures using cell impermeable dyes.  Dystrophin is also heavily involved with intracellular signaling from responses outside the cell.  Generally, proper signaling is necessary for continued cell viability.  Without it cells are usually marked for termination by immune system cells like macrophages, which seek out this kind of aberrant signaling.  DMD affected muscles are often infiltrated by these kinds of cells, which further increase the damage by initiation of an inflammatory response.

One of the hallmarks of this disease can be seen in slides of affected muscle.  Healthy cells are ensheathed by a thin membrane called the endomysium, but in diseased tissue the spaces around the muscle cells are filled increasingly with fat or fiber-like cartilaginous tissue.  This is called endomysial fibrosis.  As macrophages realize something is wrong, they infiltrate the area and initiate an immune response causing inflammation.  This further weakens the damaged cells and whittles the number of them down slowly, which accounts for the progressive weakening pathology of the disease.  The dead cells are typically replaced by this white tissue matrix.

Many hypothesized early on that gene therapy held the greatest promise to cure this disease.  After all, in order to truly cure a disease of genes, you must fix those broken genes.  Though pharmaceutical treatments reducing immune system response to the disease may improve conditions temporarily, they most likely won’t offer the long-term increase in survivability sought after for so long.  As such, many new methods and techniques have been proposed as models for new gene therapy based strategies.  Many of these have proven successful in mouse models and a few have even had some success in small clinical trials.  These cover a wide range of areas and strategies, such as using viral vectors for gene transplantation, up-regulation of suitable endogenous genes and forced expression of tailored mini genes.  Each of these has found success as a possible route towards a therapy, but each with its own drawbacks and limitations.  The master stroke, it seems, is still some time off, but even closer and within our child-like grasp at the cookie jar.

  1. Viral vectors and transgene infection

A now famous example comes from researchers at the University of London.  They worked with viral vectors to introduce a dystrophin replacement gene into muscular dystrophic mice.  Viral vectors are complicated, because they can provoke a response from the immune system, can possibly cause disease and have a limited storage capacity for any potential gene transplant.  They found a balance with the virus called Adeno-associated virus (AAV).  These viruses are able to infect many kinds of cells, can be produced without their endogenous viral genes, have never been shown to cause a human disease and also require a helper virus to replicate themselves after infection.  This combination of characteristics limits the dangers associated with infection to a degree and makes them good candidates for use in human gene therapies.

One of the main problems they faced was the small storage capacity the virus could carry.  The dystrophin gene is one of the largest known (2.4 megabases), so they had to make some kind of compromise.  They worked around this by manufacturing a mini-gene that was much smaller than the actual gene that coded for the dystrophin protein.  This was made possible by knowledge of the genomes of those affected by the disease.  A close cousin of Duchenne muscular dystrophy (DMD) is called Becker muscular dystrophy (BMD) and is characterized by a lesser severity of symptoms.  Surprisingly, the gene mutations in BMD, though sometimes ‘affecting ~ 50% of the gene itself’, affect much less critical locations than mutations in DMD.  Mapping these locations allowed them to whittle out less critical pieces of the DMD gene.  They developed a mini-gene that would produce a lesser functional, but still useful dystrophin protein, which was still able to fit within the size constraints of the viral carrier.

Once the mini-gene was completed they paired it with a transcription promoter from the cytomegalovirus to force gene transcription and production of the protein once the virus infected a cell.  They injected the virus into mice expressing a DMD phenotype and found that the mini-gene was successfully expressed in “greater than 50% of the muscle fibers 20 weeks after infection”(S).   It was found that it relieved aspects of DMD pathology, particularly rebuilding the DAP complex and improved muscle structure.  This culminated with mice test groups showing characteristics of a lesser severe form of the disease.  Furthermore, experiments showed that even with a low 20-30% level of gene expression, there was a substantial reduction in DMD pathology overall with the use of this mini gene.

2.       Increasing transcription of gene with artificial constructs

As it turns out the dystrophin protein is not the only one utilized to link the muscle to the extracellular network.  Before birth, another similar protein called utrophin is used preferentially for the same purpose, but is replaced almost entirely by dystrophin after birth. The protein exhibits 80% similarity with dystrophin and remains expressed during the course of the disease.  Researchers at the Istituto di Neurobiologia e Medicina Moleculare in Rome surmised that if preferential expression of utrophin was re-established it might provide a route to a cure or a reduction of symptoms to make it manageable.  Using an artificial transcriptional element called “Jazz”, they were able to restore muscle integrity and prevent the development of DMD in mice test groups.

It has long been known that the sudden expression of new, ‘foreign’ proteins runs the risk of causing an immune response as immune cells target these new proteins as antigens.  So just giving a person an extra supply of dystrophin won’t work as a treatment for the disease.  It was found that when a cell lacks proper functioning dystrophin, it up regulates utrophin to compensate, but the level is insufficient to prevent disease progression.  Since this protein is already expressed increased production could reduce complications caused by immune system interference.  Many previous studies have confirmed that: “Increased expression of utrophin restores plasma membrane integrity and rescues dystrophin-deficient muscle in mdx mice.”(S)

A ‘zinc finger’ is a recognition element that can interact with specific sections of DNA.  In this study, an artificial zinc finger was manufactured to correspond to a section of the promoter in the utrophin gene in both the mice and human genes.  Upon testing, they found that it was able to successfully bind to its specific DNA target sequence and increase production of utrophin expression to 1.8 times that of controls.   This increase in production also translated to a therapeutic benefit in mice test groups, showing increases in muscle size, fiber regeneration and lower serum levels of creatine kinase, a chemical identifier of muscle necrosis.

Possible benefit was further characterized by in vitro testing of muscles using electro stimulation.  Weak muscles, deficient of contractile force are a hallmark of DMD, yet those treated with the ZF ATF “Jazz” showed the opposite in excised diaphragm and extensor digitorum longus muscles.  These were able to perform longer and more sustained contractions, than diseased control groups.  Membrane integrity was also tested by staining with procion orange dye.  This fluorescent dye is usually only taken up into a cell with a leaky membrane, so it is often used to assess membrane integrity.  Sustained contractions to a muscle with a DMD phenotype would cause membrane damage and usually exhibit a greater dyed area than that of a healthy cell under the same conditions.  Muscles tested and stained under these conditions showed positive results for Jazz treated test sets, with greater dyed areas observed in the DMD cells.

3.       Antisense Oligionucleotides:

Antisense oligionucleotides are a class of nucleic acids that have also been tapped as a possible route to a DMD therapy.  In 2008, researchers at Oxford published a study testing the hypothesis that these could induce at least partial dystrophin protein expression by pushing the reading frame over in mutated muscle cells.  The idea in this technique is to use a technique called ‘exon skipping’ to push the ‘out of reading frame’ portions of the protein back into the reading frame and produce a partially functional, “becker-like” dystrophin protein.  This technique was shown to have benefit in not only mice, but also in humans in a proof of concept test in 2007.

Up until now, universal changes in expression from AOs were difficult to attain, with high percentage expression limited to skeletal muscle groups, but only limited expression in critical heart and diaphragm muscles.  This was a huge limitation to its possible use as a therapy, as “cardiomyopathy is a significant cause of morbidity and death in DMD patients” (S).  Without a corresponding effect upon heart muscle, any increase in overall muscle dystrophin expression would only exacerbate any heart conditions a DMD patient (or mouse) might have.  However, this new test was different and achieved much more favorable results in mice.  The question is: what did they do different?

It was surmised that the previous tests of AOs had limited entry into exclusive, protected environments like the heart.  They needed a tool that would allow access to these areas without destroying the gains made in earlier tests and this was achieved by conjugating the AOs to an arginine rich peptide scaffold.  Arginine is a positively charged amino acid and its use in the peptide yields an overall positive charge.  These kind of chemicals are thought to use special cell-mediated uptake systems in common with glycosaminoglycans, thus like a Trojan horse they facilitate the entry of whatever cargo they might bring.

Three weeks after injection, “between 25 and 100% of normal dystrophin levels had been restored in body-wide skeletal muscles” and “even in the diaphragm almost 25% of normal dystrophin protein was restored.”  Restoration was also seen in the heart, but not quite as high as that of skeletal muscle: “levels between 10 and 20% of that found in normal mouse heart were typically seen in all western analysis in all treated animals.” These results also corresponded with a function increase in muscle contractility and lower serum creatine kinase levels, both indicators of improved muscle ability and reduced muscle damage.

To build a tower on the sea.

Though these seem like giant steps towards a cure for one of the great diseases of man, there will undoubtedly be more questions than answers.  These are exciting times.

Every one of these people worked really hard. Please read these sources first hand (at least these):

1. “Adeno-associated virus vector gene transfer and sarcolemmal expression of a 144 kDa micro-dystrophin effectively restores the dystrophin-associated protein complex and inhibts myofibre degeneration in nude/mdx mice. Stewart A. Fabb, Dominic J. Wells, Patricia Serpente, george Dickson.  Human Molecular Genetics, 2002, vol. 11, No. 7, pgs 733-741.

2. “Expression of human full-length and minidystrophin in transgenic mdx mice: implications for gene therapy of Duchenne muscular dystrophy.” Wells DJ, Wells KE, Asante EA, Turner G, Sunada Y, Campbell KP, Walsh FS, Dickson G.  Hum Mol Genet. 1995 Aug;4(8):1245-50.

3. “The artificial gene Jazz, a transcriptional regulator of utrophin, corrects the dystrophic pathology in mdx mice.”  Maria Grazia Di Certo, Nicoletta Corbi, Georgios Strimpakos, Annalisa Onori, Siro Luvisetto, Cinzia Severini, Angelo Guglielmotti, Enrico Maria Batassa, Cinzia Pisani, Aristide Floridi, Barbara Benassi, Maurizio Fanciulli, Armando Magrelli, Elisabetta Mattei, and Claudio Passananti. Hum. Mol. Genet. (2010) 19 (5): 752-760.

4. “Cell-penetrating peptide-conjugated antisense oligionucleotides restore systemic muscle and cardiac dystrophin expression and function.”  HaiFang Yin, Hong M. Moulton, Yiqi Seow, Corinne Boyd, Jordan Boutilier, Patrick Iverson and Matthew J.A. Wood.  Hum. Mol. Genet. (2008) 17 (24): 3909-3918.

5. “Matrix metalloproteinase-9 inhibition ameliorates pathogenesis and improves skeletal muscle regeneration in muscular dystrophy.”  Hong Li, Ashwani Mittal. Denys Y. Makonchuk, Shephali Bhatnagar and Ashok Kumar.  Hum. Mol. Genet. (2009) 18 (14): 2584-2598.

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I had this conversation yesterday at a training seminar with some dude. Comments?
__________________________________________________ _________________________
Some Dude: “Bacteria don’t cause disease.”

Me: “huh.”

“Bacteria, they’re usually harmless, even helpful.”

“yeah? I know people who’ve had cholera and malaria.”

“Nah, bacteria grow into pathogenic forms when a tissue becomes too high pH, otherwise they’re harmless or helpful for digestion. It’s a conspiracy. Modern medicine has been blocking real research into the cause of disease and making money off of things like antibiotics and vaccines.”

“Wait, those are two different things.”

“No, its true. We don’t really cure anything with chemicals, our own body does the work.”

“Depends on what you mean by cure, right?”

“Time is the only thing our body needs to repair itself, the immune system does the work, not some synthetic poison. These chemicals are poison and cause cancer or worse and the industry pollutes the environment with them. Look at chemotherapy, the treatment’s worse that the disease.”

“Yeah, but people still die of disease, have always died of disease. Yeah the immune system’s great, but far from perfect. I’m still not getting were you get that bacteria don’t cause disease.”

“Exactly. Even with these chemicals, people still die. People have shown that Pasteur lied about his research. Some have shown that the pH of tissues is the real cause of disease and that too high acidity invites the bacteria to become pathogenic. Tissue pH is the root cause and the bacteria are a scapegoat.”

“OK, diphtheria, we vaccinate for it now and its not really often seen anymore in the US, but its like a plague in countries with poor water sanitation and no vaccination programs. Tissue pH? Do you know about homeostasis?”

“It’s made up, The data’s all put together by the same groups that have been illegalizing real, traditional methods for treatment. These have been known for centuries in communities: herbal remedies, acupuncture and massage that work to push the immune system to fix what’s out of balance. Now we really only have one choice.”

“Diphtheria toxin is made by the bacteria, its deadly. In a sick person or animal you can take the bacteria or even isolate just the toxin. You can take either of these and cause the same disease in a healthy animal. Part of what makes it dangerous is its method of infection: it binds to a special nuclear protein that helps produce other proteins that make life possible. Without this working properly nothing works and the cells die. Immune system cells too. This is in contrast to the pH, but I’m still not sure exactly what you mean about that. Changes in pH can denature proteins and too great a change in blood either way most definitely will kill you, but this is different. And there are bacteria that love extremes of both as well, so–“

“That’s just what they say, just parroting corporate taglines: “I’m lovin’ it”. “

Yeah, uh, well look at the time…….”

“Corporate greed will always own us, unless we change and limit our reliance on these unnecessary methods.”

“….Yeah ok…..That’s fine, I’m probably with you on limiting corporate influence, but not with the disease issue. There’s nothing special about herbal remedies, they’re full of known chemicals as well. Take aspirin, this was an herbal remedy before, but now we’ve identified what it is and how it works. Besides, herbal remedies are interventions in the same way medicines are, as far as the immune system is concerned.”

“That’s what I mean. They control us with our want to have these chemicals, they control the production of them and who can sell them. They’re even trying to control who can produce food and what kind they can produce. Its all written down in the Codex Allimentarus.”

“Well, you want to make your own aspirin? —-You could too, but I still wouldn’t buy it from you, no offense.”

“It was just willow bark extract and we can’t even make that now.”

“Yeah, but no one is stopping you from making it for yourself. Just from selling a shitty and probably dangerous product to others. A couple of years ago, there was this incident where Bayer had an impurity where cyanide was left in the product and a bunch of people died. They got sued hard and almost lost their pants. The same can’t be said of suing you though, your khakis aren’t worth that much.”

“We still have to pay them for it and are still tied to their company. It’s not needed and there’s a better way to live. Especially without them controlling us in that way. We don’t need to have a system designed to exploit us, only for them to make a profit. We shouldn’t have to live like cattle.”

“Control isn’t the word I would choose, but yeah there is a way to live without it. I haven’t made up my mind if its “better”, but we could just live with the headache. Other things aren’t the same, though. If we’re talking about certain vaccinations, like the flu then maybe, but others I wouldn’t agree, like polio or the DPT. I couldn’t risk my kid’s lives that way, too much like taking them on a drag race or…”

“Vaccinations are full of side effects they don’t tell us about, they lie about. You’re risking your kids in that way too.”

“Are you talking about the ADHD scare a while ago?”

“That’s one of them, but now the only one. They cover up the dangers of using them. Stevens-Johnson syndrome is from taking too many antibiotics, it destroys your skin, burns the skin right off.”

“CDC’s webpage offers a listing of all known side effects of the vaccines, especially the rare ones. There probably are instances where they might have covered up data or just not released it, but the fact is that side effects from them are rare. There are side effects for every medicine, though, and for the most part this information is available. I guess that part of a cover up would be that its hard to find what you’re looking for in a mountain of data, in which case that makes it a perception issue……Stevens-Johnson, Is that an autoimmune disease?

“yeah, I think so, I’m not sure what kind of–probably.”

“Those kind of diseases are where the body’s own immune system develops antibodies against a tissue. Lupus is another one, probably better known. Oh, and Rheumatic arthritis is the same. The tissues or WBCs lose the ability to tell friend from foe and attack the tissue thinking its enemy cells. But I don’t think those are caused by taking antibiotics or drugs, not usually at least. Stevens-Johnson?”

“Yeah…..Its never lupus.”

“Poor Dr. House.”

“Heh, hold on a sec–phone’s browser is a little slow….Google says fewer than 300 in the US yearly. We are becoming cyborgs! Probably that high, because of our ‘drug habit’. … But that wasn’t really your point, though— how many? It was that there are side effects?”

“Yeah, and that they do more harm than good most times with a hidden cost of making us dependent on a third party. My sister has heart problems and we found out that the medication will kill her eventually anyway.”

“Jeez, I’m sorry to hear that…”

“Us too.”

“….you still want to talk about it?”

“It’s fine.”

“I bet the Carp are going to Okinawa for training this year?”


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Back in the early days of photosynthesis, the oxygen released into the atmosphere created an oxidative environment. This converted more soluble forms of iron into the familiar orange-red, insoluble Fe+3 we see on a daily basis as rust. Iron is needed by microbes to support all sorts of biological processes, like respiration, but in the Fe+3 state it is hard to acquire and even harder to manipulate in a useful manner. Its use in cytochrome proteins of the electron transports system should underline its importance in this role. Further, in mammals the element is tightly bound to proteins such as the famous hemoglobin in red blood cells and transferrin, a mammal iron scavenger. This sequestration of iron creates a bactericidal environment inside the body and forces the invading bacteria to compete for sources of iron to fuel their biological processes.

Since it is critical to the growth and function of bacteria, especially pathogenic bacteria, it is not surprising that they have developed a variety of means to acquire this element. Some of their negative effects on a host organism directly result from this scavenging activity. Some produce proteins called hemolysins to puncture cells and release their contents, and/or produce compounds called siderophores, which are iron scavenging compounds used to leach iron and iron containing proteins from the environment.

These compounds are ubiquitous throughout the microbiological world and are usually produced under conditions of low iron concentration, like those of sterile sites in the body. While finding bacteria in cultures from skin and naso-pharyngeal swabs is not necessarily an indication of disease, finding bacteria in cultures taken from cerebral spinal fluid and blood (etc) is considered an important point in diagnosis. The skin functions as a barrier to infectious microbes, but it can be breached by pathogenic critters. Essentially, for entry into the body a microbe (or chemical) will have to cross a tissue called an epithelium. Hemolysins and cellular matrix degrading proteins are used to damage this layer and gain entry into the body and access to essential nutrients like iron. Siderophores are used to scavenge the iron from the contents of dead cells.

Siderophores vary widely in structure per species, but one thing they have in common is their preference for Fe+3. Fe+3 ions are unlike the typical organic ‘binds 4 times’ carbon atoms. Iron has a variety of oxidation states available to it and the Fe +3 state has six coordination sites arranged in the shape of an octahedron. The siderophore binding site usually accommodates this by being shaped similarly and employing atoms that also have a strong affinity for iron, specifically oxygen. Often, they employ functional groups such as catecholates and hydroxamates that can bind strongly to ions in general. These are usually arranged in such a way as to virtually guarantee that Fe+3 will be the only ion taken in by the protein. Enterobactin is a prime catecholate example of this set up, with is produced generally by enteric bacteria like Escherichia coli. Ferrichrome is another well known example of a hydroxamate compound. The binding affinity for these kind of +3 ions is very high (Ga+3 binding is also very strong), often selectively much more than +2 ions like Al+2. The fact that there is a big difference between the binding efficiency of these two oxidation states affords an efficient means of releasing the iron once it has been captured. Once the bacteria have the iron, they can reduce it to a more soluble and useful Fe+2 state.

Most bacteria have specialized receptors to accommodate these proteins, but this fact also leaves them open to attack. Bacteriophages, bacteriocins and antibiotics have been known to utilize the ferrichome transport receptors for entry into the cell. Bacteriocins are a large class of bacterially produced antibiotics used to compete with other similar strains of bacteria. Some of these have found use in medicine and agriculture to inhibit some pathogenic strains of bacteria in humans and livestock. More to the point, a new thought in antibiotic production is using these siderophore functional groups to cause their uptake by bacterial iron transport systems. The high specificity for these groups and the difficulty in changing the receptor’s structure by mutation (mutation may cause less specificity and inhibit the uptake of a vital resource) make this an interesting tactic.

By attaching a sidereophore moiety to the skeleton of an ordinary antibiotic, its potency can be increased and the effective dose lowered. In the case of attachments to the cephalosporin class of antibiotics, this effect has been studied and proven since the late 1980’s. Studies have shown that experimental catecholate cephalosporins have lower minimum inhibitory concentrations, than comparable antibiotics (frontline ceftriaxone and/or ceftazidime) making the effective dose lower than ordinary antibiotics.

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