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Loss of function in inflammation

Loss of function in inflammation



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The Wikipedia-article about Inflammation says

The five classical signs of inflammation are heat, pain, redness, swelling, and loss of function (Latin calor, dolor, rubor, tumor, and functio laesa).

What does 'loss of function' mean? And how does it occur?


TL;DR: It originally referred to abnormality in "secretion" by the inflamed tissue and later became used to mean any abnormality at all.

This fifth cardinal symptom of inflammation seems to be rather mythological as laid out in detail by Rather (1971, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1749862/?page=3). This source explains that the term seems to have been invented at some point in the 19th century without proper definition. Later authors seem to have interpreted it as basically just meaning "not functioning properly" (although this is literally the definition of unhealthy as opposed to healthy, and not specific to inflammation).

According to Rather, the original source of the confusion may have been the phrase: "… heat, redness, tumour and pain; to which should be added an alteration or suspension of the natural secretions of the part." by Macartney in 1838 (page 8 of the same document). Various versions of mentioning a fifth cardinal symptom that involved altered function made their way through pieces of writing, until it received the Latin term "functio laesa" in the late 19th century (Rather cites a piece from 1882), lending it credibility as an established concept. The passage by Macartney was later cited by Marchand in 1924, who was trying to clear up the confusion around who established the four and potentialy fifth cardinal symptoms of inflammation. Marchand rephrased it into a "fifth cardinal symptom the alteration or disappearance of normal secretion (thus a functio laesa)", trying to explain what, according to Macartney, is the fifth symptom, and adding why it could be called a "loss of function".


Usually, loss of function means some sort of limitation in physical performance. Either force, range of motion, or some other aspect of movement. In this context, loss of function can occur by at least two mechanisms:

  1. mechanical loss of function due to swelling of the tissue around the joint
  2. limitation due to pain associated with movement, part of movement, or too high load.

Inflammation

Inflammation (from Latin: inflammatio) is part of the complex biological response of body tissues to harmful stimuli, such as pathogens, damaged cells, or irritants, [1] and is a protective response involving immune cells, blood vessels, and molecular mediators. The function of inflammation is to eliminate the initial cause of cell injury, clear out necrotic cells and tissues damaged from the original insult and the inflammatory process, and initiate tissue repair.

Inflammation
The cardinal signs of inflammation include: pain, heat, redness, swelling, and loss of function. Some of these indicators can be seen here due to an allergic reaction.
SpecialtyImmunology Rheumatology
SymptomsHeat, pain, redness, swelling
ComplicationsAsthma, pneumonia, autoimmune diseases
Durationacute Few days chronic Up to many months, or years
CausesBacteria, virus

The five cardinal signs are heat, pain, redness, swelling, and loss of function (Latin calor, dolor, rubor, tumor, and functio laesa). [1] Inflammation is a generic response, and therefore it is considered as a mechanism of innate immunity, as compared to adaptive immunity, which is specific for each pathogen. [2] Too little inflammation could lead to progressive tissue destruction by the harmful stimulus (e.g. bacteria) and compromise the survival of the organism. In contrast, chronic inflammation is associated with various diseases, such as hay fever, periodontal disease, atherosclerosis, and osteoarthritis.

Inflammation can be classified as either acute or chronic. Acute inflammation is the initial response of the body to harmful stimuli and is achieved by the increased movement of plasma and leukocytes (especially granulocytes) from the blood into the injured tissues. A series of biochemical events propagates and matures the inflammatory response, involving the local vascular system, the immune system, and various cells within the injured tissue. Prolonged inflammation, known as chronic inflammation, leads to a progressive shift in the type of cells present at the site of inflammation, such as mononuclear cells, and is characterized by simultaneous destruction and healing of the tissue from the inflammatory process.

Inflammation is not a synonym for infection. Infection describes the interaction between the action of microbial invasion and the reaction of the body's inflammatory response—the two components are considered together when discussing an infection, and the word is used to imply a microbial invasive cause for the observed inflammatory reaction. Inflammation, on the other hand, describes purely the body's immunovascular response, whatever the cause may be. But because of how often the two are correlated, words ending in the suffix -itis (which refers to inflammation) are sometimes informally described as referring to infection. For example, the word urethritis strictly means only "urethral inflammation", but clinical health care providers usually discuss urethritis as a urethral infection because urethral microbial invasion is the most common cause of urethritis.

It is useful to differentiate between inflammation and infection because there are typical situations in pathology and medical diagnosis where inflammation is not driven by microbial invasion – for example, atherosclerosis, trauma, ischemia, and autoimmune diseases including type III hypersensitivity.


Loss-of-function of inositol polyphosphate-4-phosphatase reversibly increases the severity of allergic airway inflammation

Inositol polyphosphate phosphatases regulate the magnitude of phosphoinositide-3 kinase signalling output. Although inositol polyphosphate-4-phosphatase is known to regulate phosphoinositide-3 kinase signalling, little is known regarding its role in asthma pathogenesis. Here we show that modulation of inositol polyphosphate-4-phosphatase alters the severity of asthma. Allergic airway inflammation in mice led to calpain-mediated degradation of inositol polyphosphate-4-phosphatase. In allergic airway inflammation models, preventing inositol polyphosphate-4-phosphatase degradation by inhibiting calpain activity, or overexpression of inositol polyphosphate-4-phosphatase in mouse lungs, led to attenuation of the asthma phenotype. Conversely, knockdown of inositol polyphosphate-4-phosphatase severely aggravated the allergic airway inflammation and the asthma phenotype. Interestingly, inositol polyphosphate-4-phosphatase knockdown in lungs of naive mice led to spontaneous airway hyper-responsiveness, suggesting that inositol polyphosphate-4-phosphatase could be vital in maintaining the lung homeostasis. We suggest that inositol polyphosphate-4-phosphatase has an important role in modulating inflammatory response in asthma, and thus, uncover a new understanding of the complex interplay between inositol signalling and asthma, which could provide alternative strategies in asthma management.


Myeloid CFTR loss-of-function causes persistent neutrophilic inflammation in cystic fibrosis

Guoshun Wang, Department of Microbiology, Immunology and Parasitology, Louisiana State University Health Sciences Center, CSRB 607, 533 Bolivar Street, New Orleans, LA 70112, USA.

Department of Microbiology, Immunology and Parasitology, Louisiana State University Health Sciences Center, New Orleans, Louisiana, USA

Department of Microbiology, Immunology and Parasitology, Louisiana State University Health Sciences Center, New Orleans, Louisiana, USA

Department of Microbiology, Immunology and Parasitology, Louisiana State University Health Sciences Center, New Orleans, Louisiana, USA

Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan, USA

Center for Advanced Models for Translational Sciences and Therapeutics, University of Michigan Medical Center, University of Michigan Medical School, Ann Arbor, Michigan, USA

Department of Medicine, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, and Veterans Administration Medical Center, Iowa City, Iowa, USA

Department of Microbiology, Immunology and Parasitology, Louisiana State University Health Sciences Center, New Orleans, Louisiana, USA

Guoshun Wang, Department of Microbiology, Immunology and Parasitology, Louisiana State University Health Sciences Center, CSRB 607, 533 Bolivar Street, New Orleans, LA 70112, USA.

Abstract

Persistent neutrophilic inflammation is a hallmark of cystic fibrosis (CF). However, the mechanisms underlying this outstanding pathology remain incompletely understood. Here, we report that CFTR in myeloid immune cells plays a pivotal role in control of neutrophilic inflammation. Myeloid CFTR-Knockout (Mye-Cftr−/−) mice and congenic wild-type (WT) mice were challenged peritoneally with zymosan particles at different doses, creating aseptic peritonitis with varied severity. A high-dose challenge resulted in significantly higher mortality in Mye-Cftr−/− mice, indicating an intrinsic defect in host control of inflammation in mice whose myeloid cells lack CF. The low-dose challenge demonstrated an impaired resolution of inflammation in Mye-Cftr−/− mice, reflected by a significant overproduction of proinflammatory cytokines, including neutrophil chemokines MIP-2 and KC, and sustained accumulation of neutrophils. Tracing neutrophil mobilization in vivo demonstrated that myeloid CF mice recruited significantly more neutrophils than did WT mice. Pulmonary challenge with zymosan elicited exuberant inflammation in the lung and recapitulated the findings from peritoneal challenge. To determine the major type of cell that was primarily responsible for the over-recruitment of neutrophils, we purified and cultured ex vivo zymosan-elicited peritoneal neutrophils and macrophages. The CF neutrophils produced significantly more MIP-2 than did the WT counterparts, and peripheral blood neutrophils isolated from myeloid CF mice also produced significantly more MIP-2 after zymosan stimulation in vitro. These data altogether suggest that CFTR dysfunction in myeloid immune cells, especially neutrophils, leads to hyperinflammation and excessive neutrophil mobilization in the absence of infection. Thus, dysregulated inflammation secondary to abnormal or absent CFTR in myeloid cells may underlie the clinically observed neutrophilic inflammation in CF.


The Fundamental Link Between Body Weight and the Immune System

Inflammation plays a critical role in determining how we digest food, and it’s only now starting to reveal itself.

Sometimes combined with the directive move more, this mantra has a clear point. If you can’t lose weight, you are either stupid or lazy—or, probably, both. See also: Calories in, calories out.

But if things were that simple, diets would work. Middle-aged people would not suddenly start gaining weight despite eating and moving similarly year after year. No one would have to endure the presence of that one friend with the “fast metabolism” who can eat anything he wants. And who, even though he knows you’re on a diet, says through his overstuffed mouth, “I couldn’t even gain weight if I tried.”

Instead, it is becoming clear that some people’s guts are simply more efficient than others’ at extracting calories from food. When two people eat the same 3,000-calorie pizza, for example, their bodies absorb different amounts of energy. And those calorie-converting abilities can change over a person’s lifetime with age and other variables.

The question is, why? And is it possible to make changes, if a person wanted to?

If so, the solution will involve the trillions of microbes in our intestines and how they work in concert with another variable that’s just beginning to get attention. The immune system determines levels of inflammation in the gut that are constantly shaping the way we digest food—how many calories get absorbed, and how many nutrients simply pass through.

The relationship between microbes and weight gain has long been overlooked in humans, but people have known about similar effects in animals for decades. After World War II, antibiotics became affordable and abundant for the first time. Farmers began giving the drugs to their livestock—for example, to treat a milk cow’s infected udder—and noticed that animals who got antibiotics grew larger and more quickly.

This led to a flood of patent applications for antibiotic-laden foods for all sorts of livestock. In 1950, the drug company Merck filed a patent for “a method of accelerating the growth of animals” with “a novel growth-promoting factor” that was, simply, penicillin. Eli Lilly patented three new antibiotics to mix into the feed of sheep, goats, and cattle because the microbe-killing agents “increased feed efficiency.” In the ensuing decades it became standard practice to give livestock copious doses of antibiotics to make them grow faster and larger, even though no one knew why this happened, or what other effects the practice might have.

Researchers have only recently shown that these antibiotics kill off some of the microbes that occur normally in the gut and help livestock, and people, digest food. By breaking down nutrients and helping them pass through the walls of the bowel, these microbes serve as a sort of gatekeeper between what is eaten and what actually makes it into the body.

Killing them is not without consequences. Just as antibiotics are associated with faster growth in cattle, a decrease in diversity in the human microbiome is associated with obesity. As the usage of animal antibiotics exploded in the 20th century, so too did usage in humans. The rise coincides with the obesity epidemic. This could be a spurious correlation, of course—lots of things have been on the rise since the ’50s. But dismissing it entirely would require ignoring a growing body of evidence that our metabolic health is inseparable from the health of our gut microbes.

In 2006, Jeffrey Gordon, a biologist at Washington University in St. Louis, reported that the microbiomes of obese mice had something in common: Compared with their lean counterparts, the heavier mice had fewer Bacteroides and more Firmicutes species in their guts. What’s more, biochemical analyses showed that this ratio made the microbes better at “energy harvest”—essentially, extracting calories from food and passing it into the body. That is, even when mice ate the same amount and type of food, the bacterial populations meant that some developed metabolic problems, while others didn’t. Similar bacterial patterns have since been confirmed in obese humans.

What’s more, Gordon found, the microbiome associated with obesity is transferable. In 2013, his lab took gut bacteria from pairs of human twins in which only one twin was obese, then fed the samples to mice. The mice given bacteria from the obese humans quickly gained weight. The others did not.

Gut bacteria are also transferred between humans, in the form of fecal transplants, as an experimental treatment for serious infections like Clostridium difficile. In one study, obese patients who received transplants from lean donors later had healthier responses to insulin.

Short of this sort of hard reset of the microbiome, preliminary research has shown that adding even a single bacterial species to a person’s gut can alter her metabolism. In a clinical trial reported last month in the journal Nature Medicine, people who took a probiotic containing Akkermansia muciniphila—which is typically found in greater amounts in non-obese people—saw subtle metabolic improvements, including weight loss.

The study authors are not suggesting that anyone go out and buy this bacterium. But they call it a “proof of concept” for the idea that it’s possible to change a person’s microbiome in ways that have metabolic benefits.

Because leanness and obesity seem to be transmissible through the microbiome, “metabolic disease turns out to be, in some ways, like an infectious disease,” says Lora Hooper, the chair of the immunology department at the University of Texas Southwestern Medical Center. Hooper did her postdoctoral research in Gordon’s lab in St. Louis. While other researchers focused on the gut microbiome itself, she took an interest in the immune system. Specifically, she wanted to know how an inflammatory response could influence these microscopic populations, and thus be related to weight gain.

Over the past decade or so, multiple studies have shown that obese adults mount less effective immune responses to vaccinations, and that both overweight and underweight people have elevated rates of infection. But these were long assumed to be effects of obesity, not causes.

“When I started my lab there wasn’t much known about how the immune system perceives the gut microbes,” Hooper says. “A lot of people thought the gut immune system might be sort of blind to them.” To her, it was obvious that this couldn’t be the case. The human gut is host to about 100 trillion bacteria. They serve vital metabolic functions, but can quickly kill a person if they get into the bloodstream. “So clearly the immune system has got to be involved in maintaining them,” she says. It made sense to her that even subtle changes in the functioning of the immune system could influence microbial populations—and, hence, weight gain and metabolism.

This theory was borne out late last month in a paper in Science. Zac Stephens, a microbial ecologist at the University of Utah, and his colleagues had been working with mice with altered immune T cells. They noticed that over time, these mice “ballooned,” as Stephens puts it. One of his colleagues started calling them “pancakes.”

To figure out how such an immune change could cause obesity, they tested the biomes of the mice with and without the immune alteration. They found that healthy mice have plenty of bacteria from a genus called Clostridia, but few from Desulfovibrio, and that their guts let most fat pass right through. Those with an altered immune system had fewer Clostridia and more Desulfovibrio, and this microbial balance helped the gut absorb more fats from food. These mice gained more weight and exhibited signs of type 2 diabetes.

“Whether this applies in humans, we don't know,” Hooper says, “but this is a tantalizing clue.”

Mice are not humans, but their microbiomes are about as complex as our own. Reduced Clostridia and increased Desulfovibrio are seen in people with obesity and type 2 diabetes. Bacteria can reasonably be expected to function similarly in the guts of different species. But even if they don’t, this experiment is a demonstration of principle: The immune system helps control the composition of the gut microbiome.

It does so by regularly mounting low-level immune responses to keep populations of bacteria in check. “The gut is under a constant state of inflammation, so to speak—constant immune stimulation from all the microbes,” says Stephens, pushing back on the common misconception that inflammation is always bad. The role of the immune system in the gut is to maintain balance. Changes to the body’s defenses, which can happen as a result of age or illness, can cause certain species to flourish at the expense of others.

This is the interesting part to Steven Lindemann, a researcher at Purdue University who was not involved in the Utah study. He studies the effects of foods on the gut microbiome. “Although we know that, on the balance, diet is the strongest contributor to gut microbiome composition,” he said, this study suggests that when immune control of the colon breaks down, growth can become unchecked and cause problems with metabolic regulation.

Lindemann says the fact that the immune system regulates the inhabitants of the small intestine is well established. He compares the bowel wall to a customs checkpoint: The goal is to weed out bad actors and illegal cargo, but allow legitimate trade to progress as rapidly as possible. In the case of the immune-altered mice, he says, “we have a colonic border patrol that is seemingly out to lunch, allowing bad actor Desulfovibrio to bloom.”

If similar microbial changes have comparable effects in humans, it could have far-reaching implications for our diets. The very ideas of “nutritional value” and “calorie content” of food seem to vary based on the microbial population of the person eating it and, potentially, her immune status. A person’s own microbes—and those contained in any given food—would have to be considered as another ­component of the already flimsy calories-in, calories-out equation. This would also compound the challenges already facing nutrition labels.

People trying to control their weight might conclude that tinkering with their own microbiomes is the solution. This stands to fuel the already dubious and barely regulated industry of “probiotic” supplements, which has been projected to grow to $7 billion by 2025. But the answer probably won’t be so simple.

“A lot of the recent research on probiotics suggests it’s really not easy to keep and sustain new communities,” Stephens says. The immune system could explain that. “It may well be that your immune response gets ‘stuck’ at an early age based on what you’ve exposed it to. Probiotics might not be enough to change a person’s microbiome, because your immune system determined early on that certain microbes are either appropriate or inappropriate in your gut.”

Stephens says the relationship between weight and the immune system is likely to get more complicated before it gets simpler. That makes it difficult to give concrete advice. “Keeping diverse gut microbes with diverse dietary sources is probably the safest advice for now,” he says. “That will stimulate a healthy, strong immune system that can learn and regulate and do all the things it does, in ways we’re just beginning to understand.”

If all this uncertainty makes nutrition guidelines and nutrition even more inscrutable, it also stands to do some good by undermining the moralizing and simplistic character judgments often associated with body weight. Seeing obesity as a manifestation of the interplay between many systems—genetic, microbial, environmental—invites the understanding that human physiology has changed along with our relationship to the species in and around us. As these new scientific models unfold, they impugn the idea of weight as an individual character flaw, revealing it for the self-destructive myth it has always been.


Everything you need to know about inflammation

Inflammation is part of the body’s defense mechanism and plays a role in the healing process.

When the body detects an intruder, it launches a biological response to try to remove it.

The attacker could be a foreign body, such as a thorn, an irritant, or a pathogen. Pathogens include bacteria, viruses, and other organisms, which cause infections.

Sometimes, the body mistakenly perceives its own cells or tissues as harmful. This reaction can lead to autoimmune diseases, such as type 1 diabetes.

Experts believe inflammation may contribute to a wide range of chronic diseases. Examples of these are metabolic syndrome, which includes type 2 diabetes, heart disease, and obesity.

People with these conditions often have higher levels of inflammatory markers in their bodies.

In this article, find out more about why inflammation happens, its symptoms, and ways to resolve it.

Share on Pinterest A person with acute inflammation might experience pain in the affected area.

There are two main types of inflammation: acute and chronic.

Acute inflammation

An injury or illness can involve acute, or short-term, inflammation.

There are five key signs of acute inflammation:

  • Pain: This may occur continuously or only when a person touches the affected area.
  • Redness: This happens because of an increase in the blood supply to the capillaries in the area.
  • Loss of function: There may be difficulty moving a joint, breathing, sensing smell, and so on.
  • Swelling: A condition call edema can develop if fluid builds up.
  • Heat: Increased blood flow may leave the affected area warm to the touch.

These signs are not always present. Sometimes inflammation is “silent,” without symptoms. A person may also feel tired, generally unwell, and have a fever.

Symptoms of acute inflammation last a few days. Subacute inflammation lasts 2–6 weeks .

Chronic inflammation can continue for months or years. It either has or may have links to various diseases, such as:

The symptoms will depend on the disease, but they may include pain and fatigue.

Measuring inflammation

When inflammation is present in the body, there will be higher levels of substances known as biomarkers.

An example of a biomarker is C-reactive protein (CRP). If a doctor wants to test for inflammation, they may assess CRP levels.

CRP levels tend to be higher in older people and those with conditions such as cancer and obesity. Even diet and exercise may make a difference.

Inflammation happens when a physical factor triggers an immune reaction. Inflammation does not necessarily mean that there is an infection, but an infection can cause inflammation.

Acute inflammation

Acute inflammation can result from:

When the body detects damage or pathogens, the immune system triggers a number of reactions:

  • Tissues accumulate plasma proteins, leading to a buildup of fluid that results in swelling.
  • The body releases neutrophils, a type of white blood cell, or leukocyte, which move toward the affected area. Leukocytes contain molecules that can help fight pathogens.
  • Small blood vessels enlarge to enable leukocytes and plasma proteins to reach the injury site more easily.

Signs of acute inflammation can appear within hours or days, depending on the cause. In some cases, they can rapidly become severe. How they develop and how long they last will depend on the cause, which part of the body they affect, and individual factors.

Some factors and infections that can lead to acute inflammation include:

  • acute bronchitis, appendicitis and other illnesses ending in “-itis”
  • an ingrown toenail
  • a sore throat from a cold or flu
  • physical trauma or wound

Chronic inflammation

Chronic inflammation can develop if a person has:

Sensitivity: Inflammation happens when the body senses something that should not be there. Hypersensitivity to an external trigger can result in an allergy.

Exposure: Sometimes, long-term, low-level exposure to an irritant, such as an industrial chemical, can result in chronic inflammation.

Autoimmune disorders: The immune system mistakenly attacks normal healthy tissue, as in psoriasis.

Autoinflammatory diseases: A genetic factor affects the way the immune system works, as in Behçet’s disease.

Persistent acute inflammation: In some cases, a person may not fully recover from acute inflammation. Sometimes, this can lead to chronic inflammation.

Factors that may increase the risk of chronic inflammation include :

  • older age
  • a diet that is rich in unhealthful fats and added sugar
  • low sex hormones

Long-term diseases that doctors associate with inflammation include:

Inflammation plays a vital role in healing, but chronic inflammation may increase the risk of various diseases, including some cancers, rheumatoid arthritis, atherosclerosis, periodontitis, and hay fever.

The following table summarizes some key differences between acute and chronic inflammation.

AcuteChronic
CauseHarmful pathogens or tissue injury.Pathogens that the body cannot break down, including some types of viruses, foreign bodies that remain in the system, or overactive immune responses.
OnsetRapid.Slow.
DurationA few days.From months to years.
OutcomesInflammation improves, or an abscess develops or becomes chronic.Tissue death, thickening, and scarring of connective tissue.

It is essential to identify and manage inflammation and related diseases to prevent further complications.

Acute inflammation can cause pain of varying types and severity. Pain may be constant and steady, throbbing and pulsating, stabbing, or pinching.

Pain results when the buildup of fluid leads to swelling, and the swollen tissues push against sensitive nerve endings.

Other biochemical processes also occur during inflammation. They affect how nerves behave, and this can contribute to pain.

Treatment of inflammation will depend on the cause and severity. Often, there is no need for treatment.

Sometimes, however, not treating inflammation can result in life threatening symptoms.

During an allergic reaction, for example, inflammation can cause severe swelling that may close the airways, making it impossible to breathe. It is essential to have treatment if this reaction occurs.

Without treatment, some infections can enter the blood, resulting in sepsis. This is another life threatening condition that needs urgent medical treatment.

Acute inflammation

A doctor may prescribe treatment to remove the cause of inflammation, manage symptoms, or both.

For a bacterial or fungal infection, for example, they may prescribe antibiotics or antifungal treatment.

Here are some treatments specifically for treating inflammation:

Nonsteroidal anti-inflammatory drugs

Nonsteroidal anti-inflammatory drugs (NSAIDs) will not remove the cause of inflammation, but they can help relieve pain, swelling, fever, and other symptoms. They do this by countering an enzyme that contributes to inflammation.

Examples of NSAIDs include naproxen, ibuprofen, and aspirin. These are available to purchase online or over the counter. People should check first with a doctor or pharmacist to ensure they make the right choice.

People should only use NSAIDs long term if a doctor recommends them, as they can have adverse effects. Aspirin is not suitable for children.

Pain relief: Acetaminophen, including paracetamol or Tylenol, can relieve pain but does not reduce inflammation. These drugs allow the inflammation to continue its role in healing.

Corticosteroids

Corticosteroids, such as cortisol, are a type of steroid hormone. They affect various mechanisms involved in inflammation.

Corticosteroids can help manage a range of conditions, including:

They are available as pills, injections, in an inhaler, or as creams or ointments.

Long-term use of corticosteroids can be harmful. A doctor can advise on their risks and benefits.

Treatment for diseases that involve long-term inflammation will depend on the condition.

Some drugs act to repress the body’s immune reactions. These can help relieve symptoms of rheumatoid arthritis, psoriasis, and other similar autoimmune reactions. However, they can also leave a person’s body less able to fight an infection if it occurs.

People who have undergone transplant surgery also need to take immunosuppressant drugs to prevent their bodies from rejecting the new organ. They, too, need to take extra care to avoid exposure to infections.


Chemical Mediators

Injury initiates the inflammatory response, but chemical sub-stances released at the site induce the vascular changes. Foremost among these chemicals are histamine and the kinins.

Histamine is present in many tissues of the body but is concentrated in the mast cells. It is released when injury occurs and is responsible for the early changes in vasodilation and vascular permeability. Kinins increase vasodilation and vascular permeability they also attract neutrophils to the area. Prostaglandins, another group of chemical substances, are also suspected of causing increased per-meability.


“Autoinflammatory psoriasis”—genetics and biology of pustular psoriasis

Psoriasis is a chronic inflammatory skin condition that has a fairly wide range of clinical presentations. Plaque psoriasis, which is the most common manifestation of psoriasis, is located on one end of the spectrum, dominated by adaptive immune responses, whereas the rarer pustular psoriasis lies on the opposite end, dominated by innate and autoinflammatory immune responses. In recent years, genetic studies have identified six genetic variants that predispose to pustular psoriasis, and these have highlighted the role of IL-36 cytokines as central to pustular psoriasis pathogenesis. In this review, we discuss the presentation and clinical subtypes of pustular psoriasis, contribution of genetic predisposing variants, critical role of the IL-36 family of cytokines in disease pathophysiology, and treatment perspectives for pustular psoriasis. We further outline the application of appropriate mouse models for the study of pustular psoriasis and address the outstanding questions and issues related to our understanding of the mechanisms involved in pustular psoriasis.


Results

KRIT1 regulates c-Jun expression

Previously we showed that KRIT1 loss is associated with an increase in intracellular ROS levels as well as with ROS-mediated cellular dysfunctions, including a reduced ability to maintain a quiescent state [12].

A growing body of evidence suggests that the cellular response to unbalanced ROS overproduction and detoxification is primarily regulated at the level of transcription. Indeed, posttranslational modification of redox-sensitive transcription factors may provide a mechanism by which cells sense these redox changes [18].

To further characterize the functional significance of KRIT1 involvement in the maintenance of the intracellular ROS homeostasis, we analyzed the effects of KRIT1 loss on the expression of c-Jun, a redox-sensitive transcription factor known to be involved in the modulation of endothelial barrier function and angiogenesis [13,18,21,22,25,26].

As a first approach, we performed RT-qPCR and Western blotting analysis of c-Jun mRNA and protein expression levels in KRIT1 −/− (K −/− ) and wild-type (K +/+ ) MEFs, established from KRIT1 −/− and KRIT1 +/+ E8.5 mouse embryos, respectively, as well as in KRIT1 −/− MEFs re-expressing KRIT1 (K9/6) [12].

The outcomes of these experiments showed that c-Jun expression was significantly higher in K −/− than in K +/+ and K9/6 cells at both the mRNA ( Fig. 1 A) and the protein ( Fig. 1 B) levels, suggesting that KRIT1 loss leads to c-Jun upregulation.

KRIT1 regulates c-Jun expression — KRIT1 knockout and re-expression approach. KRIT1 −/− (K −/− ) and wild-type (K +/+ ) MEFs and KRIT1 −/− MEFs re-expressing KRIT1 (K9/6) were grown to confluence under standard conditions and analyzed by (A) RT-qPCR, (B, C) Western blotting, and (D) immunofluorescence as described under Material and methods. (A) RT-qPCR analysis of c-Jun mRNA expression levels. The amount of each target mRNA expressed in a sample was analyzed in triplicate using appropriate TaqMan gene expression assays (Roche) and normalized to the amounts of internal normalization control transcripts (18S rRNA). Results are expressed as relative mRNA level units referred to the average value obtained for the K −/− samples and represent the mean (±SD) of n ≥ 3 independent RT-qPCR experiments. ***P𢙀.001 versus K −/− cells. Notice that c-Jun mRNA levels are significantly higher in K −/− compared to K +/+ and K9/6 MEFs. (B) Representative Western blot analysis of the relative c-Jun, phospho-c-Jun, and KRIT1 expression levels. Tubulin (α-Tub) was used as loading control. Notice that both c-Jun and phospho-c-Jun levels are significantly higher in K −/− compared to K +/+ and K9/6 MEFs. An inverse correlation between c-Jun/phospho-c-Jun and KRIT1 protein levels is also evident. (C) Histograms showing quantitative results of Western blot analysis of the relative c-Jun, phospho-c-Jun, and KRIT1 expression levels. Optical density values are expressed as relative protein level units referred to the average value obtained for the K −/− samples and represent the mean (±SD) of n ≥ 3 independent Western blotting experiments. **P ≤ 0.01 and ***P ≤ 0.001 versus K −/− cells. Notice that differences in phospho-c-Jun levels are correlated with differences in total c-Jun levels. (D) Confocal microscopy analysis of phospho-c-Jun levels and subcellular localization in K −/− and K9/6 MEF cells. Phospho-c-Jun and nuclei were visualized with anti-phospho-c-Jun mAb coupled to Alexa Fluor 488 secondary antibody and DAPI dye, respectively. Notice that phospho-c-Jun is correctly localized to the nucleus in cells lacking KRIT1 (K −/− ) and shows enhanced levels compared to K9/6. Scale bar, 15 μm.

To evaluate whether this effect was associated with c-Jun activating phosphorylation and efficient import into the nucleus, we examined phospho-c-Jun levels and subcellular localization. Western blotting analysis of whole-cell extracts with a mAb specific for the active, phosphorylated form of c-Jun (at Ser-63 and Ser-73) (P-c-Jun) showed that phospho-c-Jun levels were always correlated with total c-Jun levels, being significantly higher in K −/− than in K +/+ and K9/6 MEF cells ( Fig. 1 B, P-c-Jun, and 1C). Furthermore, fluorescence microscopy analysis of K −/− and K9/6 MEF cells with the anti-phospho-c-Jun mAb confirmed that phospho-c-Jun levels were higher in K −/− than in K9/6 MEF cells ( Fig. 1 D, images a, b) and showed a correct nuclear localization in cells lacking KRIT1 ( Fig. 1 D, images a, c, e).

Remarkably, an inverse correlation between KRIT1 and c-Jun expression/phosphorylation levels was also observed ( Figs. 1 A𠄼), suggesting that KRIT1 plays a role in controlling c-Jun expression and activity.

To further assess this evidence, we used other cell types, including epithelial and endothelial cells, and modulated the expression of KRIT1 by two additional and complementary approaches, such as knockdown and overexpression approaches.

KRIT1 knockdown was performed in HeLa cells ( Figs. 2 A𠄾) and HUVECs ( Fig. 2 F) using two distinct KRIT1-specific siRNAs (siK655 and siK469), which induced a significant decrease in KRIT1 expression at both mRNA ( Fig. 2 A) and protein ( Figs. 2 C𠄿) levels. Notably, as detected by RT-qPCR and Western blotting assays, the siRNA-mediated knockdown of KRIT1 resulted in a significant upregulation of c-Jun mRNA ( Fig. 2 B) and protein ( Figs. 2 C𠄿) expression levels, supporting the evidence that KRIT1 downregulation causes the upregulation of c-Jun. Notably, the upregulated levels of c-Jun were again correlated with corresponding enhanced levels of the active, phosphorylated form of c-Jun ( Fig. 2 D).

KRIT1 regulates c-Jun expression — KRIT1 downregulation (siRNA) approach. (A𠄾) HeLa cells and (F) HUVECs were mock-transfected or transfected with either a KRIT1-specific siRNA (siK655 or siK469) or a negative control siRNA (siNC). 48 h posttransfection, cells were lysed and analyzed by (A, B) RT-qPCR and (C, D, F) Western blotting to assess c-Jun and KRIT1 mRNA and protein levels, respectively. 18S rRNA and tubulin (α-Tub) were used as endogenous controls for RT-qPCR normalization and Western blotting loading, respectively. Notice that the siRNA-mediated knockdown of KRIT1 results in the upregulation of c-Jun and phospho-c-Jun expression levels. Histograms show quantitative results of (A, B) RT-qPCR and (E, F) Western blot analysis of the relative c-Jun and KRIT1 mRNA and protein expression levels, respectively. mRNA levels (A, B) and optical density values of Western blot bands (E, F) are expressed as relative level units referred to the average value obtained for the mock- (E) or siNC-transfected (F) cells and represent the mean (±SD) of n = 3 independent experiments. ***P ≤ 0.001 versus mock-transfected cells.

Taken together, the KRIT1 knockout and knockdown approaches demonstrate that KRIT1 loss/downregulation is associated with a significant upregulation of c-Jun/phospho-c-Jun levels. Conversely, the forced re-expression of KRIT1 in KRIT1 −/− MEF cells to levels higher than wild-type cells ( Fig. 1 B, compare KRIT1 levels in K9/6 and K +/+ MEFs) caused a significant downregulation of c-Jun/phospho-c-Jun expression at both the protein ( Fig. 1 B) and the mRNA ( Fig. 1 A) levels, suggesting a dose-dependent inverse relationship between KRIT1 and c-Jun levels.

To provide further support to the existence of this inverse relationship, we induced KRIT1 overexpression in HeLa cells via transient transfection with a GFP-tagged KRIT1 construct [30]. Consistent with the outcomes of the alternative KRIT1 knockout and knockdown approaches described above, this third complementary approach showed that the forced upregulation of KRIT1 leads to a strong downregulation of c-Jun protein levels ( Figs. 3 A and B), clearly demonstrating that KRIT1 is able to keep c-Jun expression under strict control.

KRIT1 regulates c-Jun expression — KRIT1 overexpression approach. HeLa cells were mock-transfected or transiently transfected with a GFP-tagged KRIT1A construct. 48 h posttransfection, cells were lysed and analyzed by Western blotting with anti-c-Jun (c-Jun) and anti-GFP (GFP-KRIT1 and GFP) antibodies. Tubulin (α-Tub) was used as loading control. Notice that KRIT1 overexpression in HeLa cells results in the downregulation of c-Jun protein levels. Histograms show quantitative results of Western blotting analysis of the relative c-Jun and KRIT1 expression levels. Band optical density values are expressed as relative protein level units referred to the average value obtained for the mock-transfected cells and represent the mean (±SD) of n=3 independent Western blotting experiments. ***P𢙀.001 versus mock-transfected cells.

All together, these results suggest that KRIT1 plays a dose-dependent role in limiting c-Jun expression and activity.

ROS scavenging reverses the upregulation of c-Jun expression/phosphorylation caused by KRIT1 loss

There is clear evidence that ROS trigger c-Jun activity by both inducing c-Jun expression and activating phosphorylation [13,18].

To test whether the c-Jun upregulation observed in KRIT1 −/− MEF cells was attributable to the previously reported KRIT1 loss-dependent enhanced steady-state levels of intracellular ROS [12], we analyzed both c-Jun expression and phosphorylation in K −/− , K +/+ , and K9/6 MEF cells after cell treatment with the antioxidant NAC, which was previously demonstrated to be effective at reducing the levels of ROS in K −/− cells near to the levels of K9/6 cells and rescuing KRIT1 loss-dependent ROS-mediated molecular and cellular dysfunctions, including the upregulation of cyclin D1 and the reduced cell ability to maintain a quiescent state [12]. The outcomes of these experiments showed that treatment of KRIT1 −/− cells with NAC led to a significant reduction in both c-Jun expression and phosphorylation compared with relative levels in untreated cells ( Figs. 4 A𠄽), indicating that the enhanced c-Jun expression and phosphorylation associated with KRIT1 loss is a redox-sensitive phenomenon. Furthermore, the reduced levels of c-Jun expression and phosphorylation observed in KRIT1 −/− cells upon NAC treatment were close to levels observed in untreated wild-type cells ( Figs. 4 A and B), suggesting that the enhanced c-Jun expression and phosphorylation associated with KRIT1 loss may indeed be largely reversed by antioxidants.

ROS scavenging overcomes the upregulation of c-Jun expression/phosphorylation caused by KRIT1 loss. KRIT1 −/− (K −/− ), wild-type (K +/+ ), and KRIT1 −/− re-expressing KRIT1 (K9/6) MEFs grown to confluence were either mock-treated or treated with the ROS scavenging agent NAC (20 mM in complete medium) for 120 min at 37 ଌ. The cells were then lysed and analyzed by Western blotting with either (A) c-Jun (c-Jun) or (B) phospho-c-Jun (P-c-Jun) antibodies. Vinculin was used as loading control. Notice that c-Jun expression and phosphorylation levels in KRIT1 −/− cells treated with the ROS scavenger NAC (K −/− NAC) are significantly reduced compared with untreated KRIT1 −/− cells and close to the levels of untreated wild-type cells (K +/+ ). (C, D) Histograms showing quantitative results of Western blot analysis of c-Jun and phospho-c-Jun expression levels. Band optical density values are expressed as relative protein level units referred to the average value obtained for untreated K −/− cells and represent the mean (±SD) of n = 3 independent Western blotting experiments. **P𢙀.01 versus untreated K −/− cells.

KRIT1 overexpression prevents forced upregulation of c-Jun induced by oxidative stimuli

There is strong evidence that oxidative stress due to either exogenous oxidants or the unbalanced overproduction and detoxification of intracellular ROS, including superoxide anion (O2 •− ) and H2O2, leads to an increase in c-Jun expression and transcriptional activity [15,16,18,19,32�].

On the other hand, we showed previously that KRIT1 prevents oxidative stress-mediated cellular dysfunctions by limiting the accumulation of intracellular ROS in a dose-dependent manner [36].

In this light, we hypothesized that the expression of KRIT1 could prevent the increase in c-Jun expression levels triggered by exogenous oxidative stimuli. To test this hypothesis, c-Jun protein levels were assayed in K −/− , K +/+ , and K9/6 MEF cells either mock-treated or treated with H2O2.

Consistent with the above reported finding that KRIT1 dose-dependently regulates c-Jun steady-state levels, these levels were inversely proportional to KRIT1 expression levels in untreated MEFs ( Fig. 5 A, lanes 1, 3, and 5 5B, and 5C). However, whereas c-Jun was significantly upregulated upon H2O2 treatment in both K −/− and K +/+ MEFs, confirming that oxidative stimuli induce c-Jun upregulation [18], this did not occur in K9/6 MEFs ( Fig. 5 A, lanes 2, 4, 6, and 5B), indicating that KRIT1 overexpression prevents forced upregulation of c-Jun induced by oxidative stimuli and further suggesting that KRIT1 plays a role in protecting cells against exogenous oxidative insults.

KRIT1 overexpression prevents forced upregulation of c-Jun induced by oxidative stimuli. (A, D) KRIT1 −/− (K −/− ) and wild-type (K +/+ ) MEFs and KRIT1 −/− re-expressing KRIT1 (K9/6) MEFs grown to confluence were either mock-treated or treated with H2O2 (0.1 mM in complete medium) for 60 min at 37 ଌ. The cells were then lysed and analyzed by Western blotting with anti-c-Jun (c-Jun) and anti-KRIT1 (KRIT1) antibodies. Tubulin (α-Tub) was used as loading control. Notice that KRIT1 overexpression prevents forced upregulation of c-Jun induced by oxidative stimuli. (B, C, E) Histograms show quantitative results of Western blot analysis of the relative c-Jun (B), KRIT1 (C), and P-c-Jun (E) expression levels. Band optical density values are expressed as relative protein level units referred to the average value obtained for untreated K −/− cells and represent the mean (±SD) of n=3 independent Western blot experiments. ***P𢙀.001 versus untreated K −/− cells.

Furthermore, whereas previous FACS analysis demonstrated that KRIT1 overexpression prevents ROS enhancement in response to cell treatment with either inorganic or organic oxidants, including H2O2 and tert-butylhydroperoxide [12], Western blotting assays showed that phospho-c-Jun levels were again correlated with total c-Jun levels ( Figs. 5 A𠄾). Intriguingly, a slight downregulation of KRIT1 protein levels was also observed upon H2O2 treatment in both wild-type (K +/+ ) and KRIT1-overexpressing (K9/6) MEF cells ( Figs. 5 A, C, and D), which deserve future investigation.

C-Jun expression/phosphorylation is enhanced in CCM lesions from KRIT1 loss-of-function mutation carriers

CCM lesions are characterized by altered blood𠄻rain barrier function and increased vessel permeability due to the weakening of endothelial cell�ll junctions [6𠄸]. Interestingly, there is clear evidence that c-Jun upregulation is linked to the induction of endothelial dysfunction and vascular permeability [21,22,25,26]. In this light, we hypothesized that the KRIT1 loss-of-function-dependent upregulation of c-Jun expression and phosphorylation observed in cellular models could also occur in vivo. To address this hypothesis, we performed immunohistochemical analysis of c-Jun expression and phosphorylation levels in surgically resected human CCM specimens from patients carrying a KRIT1 loss-of-function mutation. Notably, the results of these experiments showed a significant positive staining for both total c-Jun and phospho-c-Jun in endothelial cells lining the lumen of CCM lesions compared with perilesional normal vessels, with up to 90% of positive cells in the most abnormally dilated vessels ( Fig. 6 ), clearly demonstrating that the upregulation of c-Jun caused by KRIT1 loss-of-function occurs also in vivo and suggesting a potential relationship with CCM disease.

c-Jun expression and phosphorylation are enhanced in CCM lesions from KRIT1 loss-of-function mutation carriers. Histological sections (4 µm) of paraffin-embedded CCM surgical specimens, deriving from a KRIT1 loss-of-function mutation carrier, were processed by a two-step immunohistochemical staining technique (DAKO EnVision+ System, HRP) with c-Jun and phospho-c-Jun antibodies to assess (A, B) c-Jun and (C, D) phospho-c-Jun expression in perilesional and CCM lesion vessels. Notice that many endothelial cells lining the lumen (l) of CCM vessels (B, D) were positive for c-Jun (B) and phospho-c-Jun (D), whereas neither c-Jun- (A) nor phospho-c-Jun- (C) positive staining was detected in perilesional normal vessels (A, C). Original magnification: 20×.

KRIT1 loss of function induces a ROS-dependent activation of JNK

There is evidence that the role of oxidants and oxidative stress in enhancing c-Jun expression and transcriptional activity is mediated, at least in part, by the c-Jun NH2-terminal kinase (JNK), a major upstream regulator of c-Jun. Indeed, oxidative stress induces phosphorylation and activation of JNK, facilitating its entry into the nucleus. Nuclear JNK phosphorylates c-Jun at the serine 63 and 73 regulatory sites within the N-terminal transactivation domain, enhancing its transcriptional activities [13,18].

To test whether c-Jun upregulation induced by KRIT1 loss of function was correlated with the activation of JNK, we performed Western blotting analysis of cell extracts from K −/− and K9/6 MEF cells using a monoclonal antibody specific for the active, phosphorylated form of JNK (at Thr-183 and Tyr-185) (P-JNK). The outcomes of these experiments showed that phospho-JNK levels were significantly higher in K −/− compared to K9/6 MEF cells ( Fig. 7 A, P-JNK), thus paralleling the enhanced P-c-Jun levels ( Fig. 7 A, P-c-Jun, and previous figures) in contrast, the levels of total JNK were not significantly varied ( Fig. 7 A, JNK).

KRIT1 loss of function induces a ROS-dependent activation of JNK. KRIT1 −/− (K −/− ) and KRIT1 −/− re-expressing KRIT1 (K9/6) MEFs grown to confluence were (A) left untreated or (B) either mock-treated or treated with the ROS-scavenging agent NAC (20 mM in complete medium) for 120 min at 37 ଌ. The cells were then lysed and analyzed by Western blotting as described under Material and methods. The phosphorylated JNK and total JNK were probed using anti-phospho-JNK (Thr-183/Tyr-185) antibody and anti-JNK antibody and compared to the relative P-c-Jun and KRIT1 expression levels. Total JNK and tubulin (α-Tub) served as loading controls. (A) Notice that JNK phosphorylation is significantly higher in K −/− compared to K9/6 MEFs and correlated with P-c-Jun levels. An inverse correlation between P-JNK and KRIT1 protein levels is also evident. (B) Notice that P-JNK levels in K −/− MEFs treated with the ROS scavenger NAC (K −/− NAC+) are significantly reduced compared with untreated K −/− cells (K −/− NAC−) and close to the levels of untreated K9/6 MEFs (K9/6 NAC−). A direct correlation between P-JNK and P-c-Jun and an inverse correlation between P-JNK and KRIT1 levels are also evident.

To test whether the activating phosphorylation of JNK observed in KRIT1 −/− MEF cells was ROS-dependent, we then analyzed P-JNK levels upon cell treatment with the antioxidant NAC. Indeed, cell treatment with NAC was effective at reducing both JNK and c-Jun phosphorylation ( Fig. 7 B), indicating that KRIT1 loss of function induces a ROS-dependent activation of JNK and suggesting that this activation plays an upstream regulatory role in mediating the ROS-dependent upregulation of c-Jun. However, cell treatment with an inhibitor of JNK (SP600125) rescued only partially the ROS-dependent upregulation of c-Jun induced by KRIT1 loss (data not shown), suggesting that additional regulatory factors acting upstream of c-Jun are probably involved.

KRIT1 loss of function induces downstream targets of c-Jun

Activation of c-Jun promotes induction of both proliferative and proinflammatory gene products. Notably, we previously found that KRIT1 loss of function leads to a ROS-mediated upregulation of cyclin D1 [12,37], a major c-Jun target gene involved in cell cycle progression through the G1 phase [12,37], suggesting a plausible involvement of c-Jun in the reduced cell ability to maintain a quiescent state caused by KRIT1 loss. To further extend the potential functional significance of the inverse relationship between KRIT1 expression and c-Jun expression and its oxidative stress-induced activation, we then tested c-Jun target genes known to be involved in proinflammatory responses, including COX-2, a major oxidative stress biomarker and inflammatory mediator involved in angiogenesis and vascular dysfunction [27�].

As detected by RT-qPCR and Western blotting assays, COX-2 expression was significantly higher in K −/− compared to K +/+ and K9/6 cells, at both the mRNA ( Fig. 8 A) and the protein ( Fig. 8 B) levels, suggesting that KRIT1 loss leads to COX-2 upregulation. Moreover, COX-2 protein levels were directly correlated with P-c-Jun levels ( Fig. 8 B), suggesting a potential relationship. Furthermore, immunohistochemical analysis of COX-2 expression in surgically resected human CCM specimens from patients carrying a KRIT1 loss-of-function mutation showed a significant positive staining in endothelial cells lining the lumen of CCM lesions, with most cells showing a prevalent COX-2 perinuclear/nuclear localization ( Fig. 8 C), suggesting that the upregulation of COX-2 caused by KRIT1 loss of function may occur also in vivo and pointing to a potential relationship with CCM disease.

KRIT1 loss of function causes the upregulation of COX-2. (A, B) KRIT1 −/− (K −/− ), wild-type (K +/+ ), and KRIT1 -/- re-expressing KRIT1 (K9/6) MEFs were grown to confluence under standard conditions and analyzed by (A) RT-qPCR and (B) Western blotting. (A) RT-qPCR analysis. COX-2 mRNA expression levels were analyzed in triplicate using appropriate TaqMan gene expression assays (Roche) and normalized to the amounts of internal normalization control transcripts (18S rRNA). Results are expressed as relative mRNA level units referred to the average value obtained for the K −/− samples and represent the mean (±SD) of n=3 independent RT-qPCR experiments. ***P𢙀.001 versus K −/− cells. (B) Representative Western blot analysis. COX-2 levels in cell lysates were analyzed by Western blotting with an anti-COX-2 mAb and compared to the relative P-c-Jun and KRIT1 levels. Tubulin (α-Tub) was used as loading control. Notice that KRIT1 loss of function reproduced in K −/− cells caused a significant upregulation of COX-2 expression at both mRNA and protein levels, which was completely rescued by the re-expression of KRIT1 (K9/6). It is also evident that COX-2 protein levels are directly and inversely correlated with P-c-Jun and KRIT1 protein levels, respectively. (C) Immunohistochemical analysis of paraffin-embedded human cerebral cavernous malformations with anti-COX-2 antibodies. Cavernous malformation tissue was collected from a CCM1 mutation carrier with familial disease at the time of surgical resection under an approved institutional review board protocol. Notice that many endothelial cells lining the lumen (l) of CCM vessels were positive for COX-2.

Whereas further studies based on c-Jun dominant negative inhibition and RNAi-mediated knockdown are required to verify the putative direct relationship between the KRIT1 loss-dependent upregulation of c-Jun and its target genes, including but not limited to cyclin D1 and COX2, the findings that KRIT1 plays a role in regulating distinct proteins involved in oxidative stress responses open novel avenues for future investigations aimed at better defining the molecular mechanisms of CCM pathogenesis.