ok, who's hasselbeck?

ok, this was awesome, and i loved inception.

solving amyloidosis

First described by none other than Rudolph Virchow (pictured, rocking a killer beard) in 1853 in reference to abnormal extracellular material found in the liver on autopsy, amyloidosis refers to the accumulation of misfolded extracellular proteins that are deposited in tissue as bundles of beta-sheet, fibrillar insoluble aggregates and ultimately result in end-organ pathology. It is seen in numerous diseases which can be stratified as follows:

Diseases in which amyloid accumulation is critical to pathogenesis: Alzheimer's disease (APP accumulation in the brain), Creutzfeldt-Jacob Disease (PrP accumulation in the brain), dialysis-associated amyloidosis (beta-2 microglobulin accumulation in the joint spaces), and senile/systemic amyloidosis (transthyretin accumulation in the brain, heart, and kidney).

Diseases in which amyloidosis is a disease-modifying side effect: multiple myeloma (accumulation of immunoglobulin light chain in kidneys and peripheral nerves), and reactive amyloidosis of chronic inflammatory disease (amyloid A accumulation).

How does amyloidosis occur? When proteins are initially translated from mRNA, their three-dimensional structure is determined by specific interactions of the amino acid side chains. With the assistance of chaperone proteins, newly produced polypeptides assemble into the secondary, tertiary, and quatenary structure at the lowest energy configuration. However, certain proteins may have more than one low-energy state, depending on the protein microenvironment: the properly assembled, functional assembly, and a misfolded assembly driven by hydrogen bonding between the amide and carbonyl groups of the main amino acid chain, which is facilitated when the polypeptides are layered in antiparallel linear sheets in which the N and C termini are oriented in opposite directions. Therefore, there are 2 major issues of concern:

1) What type of proteins are susceptible to misfolding? Certain intrinsically susceptible proteins such as transthyretin, proteins that acquire a susceptible point mutation, as seen in hereditary amyloidosis, and proteins that achieve saturating concentrations in serum, such as beta-2 microglobulin in dialysis patients.

2) What conditions support pathologic misfolding of the aforementioned proteins? Conditions that interfere with weak peptide interactions (low pH, accumulation of free radical species, high temperature).

It is important (foreshadowing) to note that amyloid accumulations are not composed simply of these fibrillar beta-sheets. A critical component of amyloid is SAP, a glycoprotein that binds the common conformation of amyloid fibrils and which is protected against proteolysis, making amyloid fibrils resistant to degradation.

The presenting symptoms of amyloidosis depend on the primary tissue in which amyloid is deposited, despite the fact that in patients with amyloidosis, autopsy routinely demonstrates some degree of deposition in every organ system. Perhaps because of this, the most common initial presenting symptoms (although not that which leads to diagnosis) in patients with amyloidosis are fatigue and weight loss. End-organ damage in amyloidosis is primary caused by tissue distortion, although there may a role for an inflammatory response to deposited amyloid as well. The most common organs affected are the heart and kidneys, but other organs can be affected as well:

Renal amyloidosis often presents with the nephrotic syndrome, with renal failure, proteinuria, hyperlipidemia, and hypoalbuminemia.

Cardiac amyloidosis often presents as a restrictive cardiomyopathy with signs of right-sided heart failure (elevated jugular venous pulses, hepatic congestion, and peripheral edema). EKG often shows low voltage, and echocardiography often reveals concentric hypertrophy with elevated filling pressures. Patients may present with an MI despite no coronary artery disease. Amyloid infiltration often has a predilection for the cardiac conduction system resulting in bradyarrhythmias and even asystole.

Neuropathies are usually autonomic (orthostatic hypotension) or sensory (carpal tunnel or symmetric, distal, painful sensory neuropathies).

GI symptoms are often secondary to enteric dysfunction, resulting in constipation and/or diarrhea, but diffuse infiltration can often result in malabsorption

Vascular infiltration results in friable blood vessels, presenting as easy bruising, with the characteristic "raccoon eyes" (spontaneous periorbital bruising following rubbing of the eyes or nose-blowing).

Soft tissue involvement results in macroglossia, submandibular enlargement.

Diagnosis is classically obtained by either involved organ or abdominal fat pad biopsy. Following Congo-Red staining of tissue, apple-green birefringence under polarized light is pathognomonic. Once found, patients should be evaluated for occult plasma cell dyscrasias, transthyretin mutations, or collagen vascular disease.

Treatment of amyloidosis has been notoriously difficult, and has revolved around reducing the rate of synthesis of the amyloidogenic protein. This is based on the idea that slow resorption of amyloid components by tissue-resident macrophages is opposed by constant renewal of amyloid protein. This has proven to be notoriously difficult, other than cases in which the culprit disease (multiple myeloma, collagen vascular disease) can be treated. Vaccination against the amyloidogenic variants of A-beta peptides in Alzheimer's Disease showed some promise in early mouse studies, but has been largely disappointing in human clinical trials. Because of this, overall prognosis of patients with amyloidosis has been overwhelmingly poor.

So why have I wasted this much breath on this topic? Because there's been a breakthrough, that's why!!

We salute you, Mark Pepys' lab at the University College London! Because you did something awesome. You realized that SAP (remember??) is critical for the stability of amyloid fibrils in vivo. You then developed a proline-based compound, CPHPC (the full name is too long), which effectively binds and depletes circulating human SAP (a component of C-reactive protein) and leaves only SAP which is bound to amyloid. You then treated this residual sap with a specific IgG antibody. Right off the bat, you showed that CPHPC followed by anti-SAP treatment significantly reduced tissue amyloid burden without any significant adverse effects in mice with human SAP-induced amyloid A-driven angiopathy (a mouse model for inflammatory amyloidosis). You then carefully demonstrated the mechanism by which this occurs:

- 24 hours after antibody treatment, you show mononuclear, primarily macrophage infiltration of amyloid accumulations, as indicated by F4/80 staining in (b).
- 48 hours after treatment, you show coalescence of macrophages into multinucleated giant cells containing amyloid within endocytic compartments, as seen in (e).
- 96 hours after, you show that diminished, fragmented amyloid fibrils are largely contained within multinucleate giant cells, as seen in (d).
-Finally, you demonstrate that this process is not only macrophage dependent, but also Fc and C3 dependent, providing a step-by-step mechanism for complement mediated clearance.

What else is there to say? With a humanized monoclonal anti-SAP antibody already being tested, this work may ultimately constitute a paradigm shift in the treatment of this disease.

Stay tuned for my next post, about hormones! The lady kinds!

the NFL

Why don't we start with a few deep thoughts from NFL Pittsburgh Steeler linebacker James Harrison, following a particularly vicious hit on the Cleveland Browns' receiver Josh Cribbs:

The crown of Harrison's helmet slammed into the left side of Cribbs' helmet as the receiver was running a Wildcat formation play, causing Cribbs to crumple face-first into the turf. He appeared to be momentarily knocked out. Because Cribbs was a runner, such helmet-to-helmet contact is permissible. "I thought Cribbs was asleep," Harrison said. "A hit like that geeks you up, especially when you find out the guy is not really hurt, he's just sleeping. He's knocked out but he's going to be OK."

Awesome. Now, my first response upon reading this was to immediately Wikipedia James Harrison, because I was absolutely dying to find out the extent of his higher education. And, oh, man, I was so not disappointed. I had forgotten that this was the same guy who in 2009, after the Steelers won the Super Bowl, was the lone member to decline an invitation to the White House. At the time, he said, "This is how I feel — if you want to see the Pittsburgh Steelers, invite us when we don't win the Super Bowl. As far as I'm concerned, he [Obama] would've invited Arizona if they had won." Which, I suppose, is true.

Despite astute observations such as those above, Harrison somehow missed the plethora of medical studies linking serial head trauma to neurocognitive impairment. Many of these studies were published in the Journal of the American Medical Association, including the first description in 1928 by a pathologist from New Jersey, Harrison (ha!) Martland, who noted a "punch-drunk" personality change in long-term boxers. Many years later, a seminal report in JAMA from 1984 (pictured) demonstrated a clear association between boxers, cerebral atrophy on CT, and impaired neurocognitive and neuropsychiatric testing with an emphasis on poor memory performance.

JAMA continued to build on these reports, and began extending their observations to another sport rife with cerebral trauma: American football. In 2003, they published a report on collegiate football players that demonstrated an additive effect to head trauma; with progressive concussions, recovery time to full neurological function increased exponentially. This followed strong experimental data in which mice subjected to repeated mild trauma suffered progressive neurological damage as a result (J Neurosurg, 2001 Nov;95(5):859-70). Follow-up studies suggested that football players with 3 or more concussions over their careers were five times more likely to show signs of mild cognitive impairment, a frequent precursor of Alzeimer's Disease.

This correlative data was provided with a histopathologic correlative in 2005 when analysis of the brain of a recently deceased football player revealed "chronic traumatic encephalopathy with many diffuse amyloid plaques as well as sparse neurofibrillary tangles and tau-positive neuritic threads in neocortical areas. " As reported in the New York Times, five subsequent autopsies of NFL veterans who died between age 36 and 50 all revealed evidence of so-named "chronic traumatic encephalopathy," with tau and amyloid deposition that was otherwise never seen in brains of people this young. As the neuropathologist Daniel Perl from Mount Sinai School of Medicine noted,

"Something is clearly abnormal in these athletes' brains...in the most extreme cases the extent of tau pathology is just unbelievable."

An excellent review in The New England Journal of Medicine summarizes the pathophysiologic mechanism as follows: following head trauma, sudden movement produces rotational injury, damaging the long axons that connect different brain regions. This results in pathologic deposition of soluble amyloid-beta peptide as soon as two hours after injury. Ultimately, this results in accelerated formation neurofibrillary tangles seen generally in older patients with dementia as well as these patients.

This and countless other reports must have been part of the inspiration for a furious piece written by Malcolm Gladwell last year in the New Yorker in which he wondered if football and dogfighting were all that different. Gladwell speaks of the countless circulating stories of bizarre and ultimately self-destructive behavior of football players following retirement:

Mike Webster, the longtime Pittsburgh Steeler and one of the greatest players in N.F.L. history, ended his life a recluse, sleeping on the floor of the Pittsburgh Amtrak station. Another former Pittsburgh Steeler, Terry Long, drifted into chaos and killed himself four years ago by drinking antifreeze. Andre Waters, a former defensive back for the Philadelphia Eagles, sank into depression and pleaded with his girlfriend—“I need help, somebody help me”—before shooting himself in the head.

“There is something wrong with this group as a cohort,” Dr. Omalu [the pathologist at U. Pittsburgh who performed the first autopsy of an NFL player showing tauopathy] says. “They forget things. They have slurred speech. I have had an N.F.L. player come up to me at a funeral and tell me he can’t find his way home. I have wives who call me and say, ‘My husband was a very good man. Now he drinks all the time. I don’t know why his behavior changed.’ I have wives call me and say, ‘My husband was a nice guy. Now he’s getting abusive.’ I had someone call me and say, ‘My husband went back to law school after football and became a lawyer. Now he can’t do his job. People are suing him.’”

Gladwell goes on to note the data demonstrating the clear risk posed to football players due not only to their full concussions, but countless subconcussive head injuries. Ultimately, he, I think rightly, lays the blame at the feet of the NFL as an organization, which, despite claims to the contrary, applauds the tenacity of hard-hitting players such as Harrison, Ray Lewis, or Brian Dawkins and lionizes "warriors" who "play through injuries" and "never miss a start." A culture such as this, in which players are taught that approval (and salary) comes with masking or minimizing injury, betrays the best interest of the players. Gladwell concludes with anecdote of a young player during practice whose concussive symptoms are so severe that he is losing consciousness while sitting in a tub of ice-cold water, yet insists on returning to practice. He notes:

That moment in the cold tub represented a betrayal of trust. He had taken the hit on behalf of his team. He was then left to pass out in the cold tub, and to deal—ten and twenty years down the road—with the consequences. No amount of money or assurances about risk freely assumed can change the fact that, in this moment, an essential bond had been broken. What football must confront, in the end, is not just the problem of injuries or scientific findings. It is the fact that there is something profoundly awry in the relationship between the players and the game.

The mixed nature of the messages sent by the league could not be clearer than in the case I first mentioned, in which James Harrison was not flagged for the hit he put on Josh Cribbs, yet is being fined $75,000 for his actions. The league wants to permit the type of violent play for which fans clamor, yet have the appearance of chastising such choices. It's reminiscent of Obama's waffling on the "right versus wisdom" of the mosque at Ground Zero. It's no wonder that Harrison, 2008's NFL Defensive Player of the Year, now being subtly undermined as a dirty player, is considering retirement. He's the cuckhold, not the true villain.

dementia pugilistica

Forthcoming.

neutrophil chemotaxis

Neutrophils are the first of the innate immune system to arrive on the scene following tissue injury. This tremendous paper by McDonald and colleagues elegantly delineates the three-step process by which this happens.

First, using a thermal injury model that causes tissue necrosis (marked by propridium iodide, red), a labelled, soluble dextran that marks tissue vasculature (blue), the authors show that neutrophils hone expertly to the site of tissue necrosis primary via an intravascular, rather than an extravascular path (aka, they follow tracks marked by vasculature delineated in blue). The authors note that neutrophils take the longest possible intravascular tract, even at the expense of taking the shortest track; they hypothesize that this reduces potential collateral damage that could be exerted by neutrophils trafficking through healthy tissue on the way to a damaged focus.

The authors also show that tissue necrosis results in unique extracellular release of ATP, which is normally restricted to within cells. Extracellular ATP then activates the receptor P2X7-R on tissue-resident macrophages and stimulates them to produce pro-inflammatory cytokines such as IL-1B. Local endothelial cells then respond to this inflammation and upregulate expression of the integrin molecule ICAM-1; it is expression of these integrins which causes neutrophils to stick to the endothelium proximal to the necrotic focus; however, neither ATP nor IL-1B is responsible for actually attracting neutrophils to areas of necrosis, only adhesion at the focus.

Second, the authors then map out the pathway by which neutrophils arrive at the necrotic focus. The inflammation generated by tissue-resident macrophages drives expression of not only ICAM-1 but also the chemokinetic ligand MIP-2 (macrophage inflammatory protein 2), otherwise known as CXCL2. The authors clearly demonstrate that, unlike ICAM-1, MIP-2 is expressed on endothelial cells in a gradient that serves as a potent chemokinetic signal for neutrophils to the edge of the necrotic focus. Interestingly, however, the authors note that endothelial cells within 150 microns of the necrotic center do not express MIP-2, and neutrophils migrate in a pattern that is independent of exogenous MIP-2 expression in the necrotic center, suggesting that an overriding signal drives neutrophil chemotaxis within the proximal 150 microns of the necrotic focus.

Once neutrophils get within 150 microns of the necrotic focus, then what happens? The authors finally demonstrate that necrotic cells release so-called damage-associated molecular patterns (DAMPs), which are generated from fragments of cellular mitochondria. DAMPs share significant homology with pathogen-associated molecular patterns (PAMPs), which are derived from bacterial fragments; this should come as no surprise, as bacteria are thought to be evolutionary precursors of eukaryotic mitochondria. These DAMPs activate signaling through formylated peptide receptors on neutrophils, which serve as an overriding signal to drive the remaining migration of neutrophils to the necrotic focus.

i gotta start posting again.

And im going to start by reading the above article in the latest issue of Science and commenting on it. Tonight.

Potential legal trouble

Looks like this Lipinski dude got the drop on me.