13.2: Autoimmune Disorders - Biology

13.2: Autoimmune Disorders - Biology

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skills to develop

  • Explain why autoimmune disorders develop
  • Provide a few examples of organ-specific and systemic autoimmune diseases

In 1970, artist Walt Kelly developed a poster promoting Earth Day, featuring a character from Pogo, his daily newspaper comic strip. In the poster, Pogo looks out across a litter-strewn forest and says wryly, “We have met the enemy and he is us.” Pogo was not talking about the human immune system, but he very well could have been. Although the immune system protects the body by attacking invading “enemies” (pathogens), in some cases, the immune system can mistakenly identify the body’s own cells as the enemy, resulting in autoimmune disease.

Autoimmune diseases are those in which the body is attacked by its own specific adaptive immune response. In normal, healthy states, the immune system induces tolerance, which is a lack of an anti-self immune response. However, with autoimmunity, there is a loss of immune tolerance, and the mechanisms responsible for autoimmune diseases include type II, III, and IV hypersensitivity reactions. Autoimmune diseases can have a variety of mixed symptoms that flare up and disappear, making diagnosis difficult.

The causes of autoimmune disease are a combination of the individual's genetic makeup and the effect of environmental influences, such as sunlight, infections, medications, and environmental chemicals. However, the vagueness of this list reflects our poor understanding of the etiology of these diseases. Except in a very few specific diseases, the initiation event(s) of most autoimmune states has not been fully characterized.

There are several possible causes for the origin of autoimmune diseases and autoimmunity is likely due to several factors. Evidence now suggests that regulatory T and B cells play an essential role in the maintenance of tolerance and prevention of autoimmune responses. The regulatory T cells are especially important for inhibiting autoreactive T cells that are not eliminated during thymic selection and escape the thymus (see T Lymphocytes and Cellular Immunity). In addition, antigen mimicry between pathogen antigens and our own self antigens can lead to cross-reactivity and autoimmunity. Hidden self-antigens may become exposed because of trauma, drug interactions, or disease states, and trigger an autoimmune response. All of these factors could contribute to autoimmunity. Ultimately, damage to tissues and organs in the autoimmune disease state comes as a result of inflammatory responses that are inappropriate; therefore, treatment often includes immunosuppressive drugs and corticosteroids.

Organ-Specific Autoimmune Diseases

Some autoimmune diseases are considered organ specific, meaning that the immune system targets specific organs or tissues. Examples of organ-specific autoimmune diseases include celiac disease, Graves disease, Hashimoto thyroiditis, type I diabetes mellitus, and Addison disease.

Celiac Disease

Celiac disease is largely a disease of the small intestine, although other organs may be affected. People in their 30s and 40s, and children are most commonly affected, but celiac disease can begin at any age. It results from a reaction to proteins, commonly called gluten, found mainly in wheat, barley, rye, and some other grains. The disease has several genetic causes (predispositions) and poorly understood environmental influences. On exposure to gluten, the body produces various autoantibodies and an inflammatory response. The inflammatory response in the small intestine leads to a reduction in the depth of the microvilli of the mucosa, which hinders absorption and can lead to weight loss and anemia. The disease is also characterized by diarrhea and abdominal pain, symptoms that are often misdiagnosed as irritable bowel syndrome.

Diagnosis of celiac disease is accomplished from serological tests for the presence of primarily IgA antibodies to components of gluten, the transglutinaminase enzyme, and autoantibodies to endomysium, a connective tissue surrounding muscle fibers. Serological tests are typically followed up with endoscopy and biopsy of the duodenal mucosa. Serological screening surveys have found about 1% of individuals in the United Kingdom are positive even though they do not all display symptoms.1 This early recognition allows for more careful monitoring and prevention of severe disease.

Celiac disease is treated with complete removal of gluten-containing foods from the diet, which results in improved symptoms and reduced risk of complications. Other theoretical approaches include breeding grains that do not contain the immunologically reactive components or developing dietary supplements that contain enzymes that break down the protein components that cause the immune response.2

Disorders of the Thyroid

Graves disease is the most common cause of hyperthyroidism in the United States. Symptoms of Graves disease result from the production of thyroid-stimulating immunoglobulin (TSI) also called TSH-receptor antibody. TSI targets and binds to the receptor for thyroid stimulating hormone (TSH), which is naturally produced by the pituitary gland. TSI may cause conflicting symptoms because it may stimulate the thyroid to make too much thyroid hormone or block thyroid hormone production entirely, making diagnosis more difficult. Signs and symptoms of Graves disease include heat intolerance, rapid and irregular heartbeat, weight loss, goiter (a swollen thyroid gland, protruding under the skin of the throat [Figure (PageIndex{1})) and exophthalmia (bulging eyes) often referred to as Graves ophthalmopathy (Figure (PageIndex{2})).

Figure (PageIndex{1}): Goiter, a hypertrophy of the thyroid, is a symptom of Graves disease and Hashimoto thyroiditis.

The most common cause of hypothyroidism in the United States is Hashimoto thyroiditis, also called chronic lymphocytic thyroiditis. Patients with Hashimoto thyroiditis often develop a spectrum of different diseases because they are more likely to develop additional autoimmune diseases such as Addison disease (discussed later in this section), type 1 diabetes, rheumatoid arthritis, and celiac disease. Hashimoto thyroiditis is a TH1 cell-mediated disease that occurs when the thyroid gland is attacked by cytotoxic lymphocytes, macrophages, and autoantibodies. This autoimmune response leads to numerous symptoms that include goiter (Figure (PageIndex{1})), cold intolerance, muscle weakness, painful and stiff joints, depression, and memory loss.

Figure (PageIndex{2}): Exophthalmia, or Graves ophthalmopathy, is a sign of Graves disease. (credit: modification of work by Jonathan Trobe, University of Michigan Kellogg Eye Center)

Type 1 Diabetes

Juvenile diabetes, or type 1 diabetes mellitus, is usually diagnosed in children and young adults. It is a T-cell-dependent autoimmune disease characterized by the selective destruction of the β cells of the islets of Langerhans in the pancreas by CD4 TH1-mediated CD8 T cells, anti-β-cell antibodies, and macrophage activity. There is also evidence that viral infections can have either a potentiating or inhibitory role in the development of type 1 diabetes (T1D) mellitus. The destruction of the β cells causes a lack of insulin production by the pancreas. In T1D, β-cell destruction may take place over several years, but symptoms of hyperglycemia, extreme increase in thirst and urination, weight loss, and extreme fatigue usually have a sudden onset, and diagnosis usually does not occur until most β cells have already been destroyed.

Autoimmune Addison Disease

Destruction of the adrenal glands (the glands lying above the kidneys that produce glucocorticoids, mineralocorticoids, and sex steroids) is the cause of Addison disease, also called primary adrenal insufficiency (PAI). Today, up to 80% of Addison disease cases are diagnosed as autoimmune Addison disease (AAD), which is caused by an autoimmune response to adrenal tissues disrupting adrenal function. Disruption of adrenal function causes impaired metabolic processes that require normal steroid hormone levels, causing signs and symptoms throughout the body. There is evidence that both humoral and CD4 TH1-driven CD8 T-cell–mediated immune mechanisms are directed at the adrenal cortex in AAD. There is also evidence that the autoimmune response is associated with autoimmune destruction of other endocrine glands as well, such as the pancreas and thyroid, conditions collectively referred to as autoimmune polyendocrine syndromes (APS). In up to 80% of patients with AAD, antibodies are produced to three enzymes involved in steroid synthesis: 21-hydroxylase (21-OH), 17α-hydroxylase, and cholesterol side-chain–cleaving enzyme.3The most common autoantibody found in AAD is to 21-OH, and antibodies to any of the key enzymes for steroid production are diagnostic for AAD. The adrenal cortex cells are targeted, destroyed, and replaced with fibrous tissue by immune-mediated inflammation. In some patients, at least 90% of the adrenal cortex is destroyed before symptoms become diagnostic.

Symptoms of AAD include weakness, nausea, decreased appetite, weight loss, hyperpigmentation (Figure (PageIndex{3})), hyperkalemia (elevated blood potassium levels), hyponatremia (decreased blood sodium levels), hypoglycemia(decreased levels of blood sugar), hypotension (decreased blood pressure), anemia, lymphocytosis (decreased levels of white blood cells), and fatigue. Under extreme stress, such as surgery, accidental trauma, or infection, patients with AAD may experience an adrenal crisis that causes the patient to vomit, experience abdominal pain, back or leg cramps, and even severe hypotension leading to shock.

Figure (PageIndex{3}): Hyperpigmentation is a sign of Addison disease. (credit: modification of work by Petros Perros)

Exercise (PageIndex{1})

  1. What are the names of autoimmune diseases that interfere with hormone gland function?
  2. Describe how the mechanisms of Graves disease and Hashimoto thyroiditis differ.
  3. Name the cells that are destroyed in type 1 diabetes mellitus and describe the result.

Systemic Autoimmune Diseases

Whereas organ-specific autoimmune diseases target specific organs or tissues, systemic autoimmune diseases are more generalized, targeting multiple organs or tissues throughout the body. Examples of systemic autoimmune diseases include multiple sclerosis, myasthenia gravis, psoriasis, rheumatoid arthritis, and systemic lupus erythematosus.

Multiple Sclerosis

Multiple sclerosis (MS) is an autoimmune central nervous system disease that affects the brain and spinal cord. Lesions in multiple locations within the central nervous system are a hallmark of multiple sclerosis and are caused by infiltration of immune cells across the blood-brain barrier. The immune cells include T cells that promote inflammation, demyelination, and neuron degeneration, all of which disrupt neuronal signaling. Symptoms of MS include visual disturbances; muscle weakness; difficulty with coordination and balance; sensations such as numbness, prickling, or “pins and needles”; and cognitive and memory problems.

Myasthenia Gravis

Autoantibodies directed against acetylcholine receptors (AChRs) in the synaptic cleft of neuromuscular junctions lead to myasthenia gravis (Figure (PageIndex{4})). Anti-AChR antibodies are high-affinity IgGs and their synthesis requires activated CD4 T cells to interact with and stimulate B cells. Once produced, the anti-AChR antibodies affect neuromuscular transmission by at least three mechanisms:

Exercise (PageIndex{2})

  • Complement binding and activation at the neuromuscular junction
  • Accelerated AChR endocytosis of molecules cross-linked by antibodies
  • Functional AChR blocking, which prevents normal acetylcholine attachment to, and activation of, AChR

Regardless of the mechanism, the effect of anti-AChR is extreme muscle weakness and potentially death through respiratory arrest in severe cases.

Figure (PageIndex{4}): Myasthenia gravis and impaired muscle contraction. (a) Normal release of the neurotransmitter acetylcholine stimulates muscle contraction. (b) In myasthenia gravis, autoantibodies block the receptors for acetylcholine (AChr) on muscle cells, resulting in paralysis.


Psoriasis is a skin disease that causes itchy or sore patches of thick, red skin with silvery scales on elbows, knees, scalp, back, face, palms, feet, and sometimes other areas. Some individuals with psoriasis also get a form of arthritis called psoriatic arthritis, in which the joints can become inflamed. Psoriasis results from the complex interplay between keratinocytes, dendritic cells, and T cells, and the cytokines produced by these various cells. In a process called cell turnover, skin cells that grow deep in the skin rise to the surface. Normally, this process takes a month. In psoriasis, as a result of cytokine activation, cell turnover happens in just a few days. The thick inflamed patches of skin that are characteristic of psoriasis develop because the skin cells rise too fast.

Rheumatoid Arthritis

The most common chronic inflammatory joint disease is rheumatoid arthritis (RA) (Figure (PageIndex{5})) and it is still a major medical challenge because of unsolved questions related to the environmental and genetic causes of the disease. RA involves type III hypersensitivity reactions and the activation of CD4 T cells, resulting in chronic release of the inflammatory cytokines IL-1, IL-6, and tumor necrosis factor-α (TNF-α). The activated CD4 T cells also stimulate the production of rheumatoid factor (RF) antibodies and anticyclic citrullinated peptide antibodies (anti-CCP) that form immune complexes. Increased levels of acute-phase proteins, such as C-reactive protein (CRP), are also produced as part of the inflammatory process and participate in complement fixation with the antibodies on the immune complexes. The formation of immune complexes and reaction to the immune factors cause an inflammatory process in joints, particularly in the hands, feet, and legs. Diagnosis of RA is based on elevated levels of RF, anti-CCP, quantitative CRP, and the erythrocyte sedimentation rate (ESR) (modified Westergren). In addition, radiographs, ultrasound, or magnetic resonance imaging scans can identify joint damage, such as erosions, a loss of bone within the joint, and narrowing of joint space.

Figure (PageIndex{5}): The radiograph (left) and photograph (right) show damage to the hands typical of rheumatoid arthritis. (credit right: modification of work by “handarmdoc”/Flickr)

Systemic Lupus Erythematosus

The damage and pathology of systemic lupus erythematosus (SLE) is caused by type III hypersensitivity reactions. Autoantibodies produced in SLE are directed against nuclear and cytoplasmic proteins. Anti-nuclear antibodies (ANAs) are present in more than 95% of patients with SLE,4 with additional autoantibodies including anti-double–stranded DNA (ds-DNA) and anti-Sm antibodies (antibodies to small nuclear ribonucleoprotein). Anti-ds-DNA and anti-Sm antibodies are unique to patients with SLE; thus, their presence is included in the classification criteria of SLE. Cellular interaction with autoantibodies leads to nuclear and cellular destruction, with components released after cell death leading to the formation of immune complexes.

Because autoantibodies in SLE can target a wide variety of cells, symptoms of SLE can occur in many body locations. However, the most common symptoms include fatigue, fever with no other cause, hair loss, and a sunlight-sensitive "butterfly" or wolf-mask (lupus) rash that is found in about 50% of people with SLE (Figure (PageIndex{6})). The rash is most often seen over the cheeks and bridge of the nose, but can be widespread. Other symptoms may appear depending on affected areas. The joints may be affected, leading to arthritis of the fingers, hands, wrists, and knees. Effects on the brain and nervous system can lead to headaches, numbness, tingling, seizures, vision problems, and personality changes. There may also be abdominal pain, nausea, vomiting, arrhythmias, shortness of breath, and blood in the sputum. Effects on the skin can lead to additional areas of skin lesions, and vasoconstriction can cause color changes in the fingers when they are cold (Raynaud phenomenon). Effects on the kidneys can lead to edema in the legs and weight gain. A diagnosis of SLE depends on identification of four of 11 of the most common symptoms and confirmed production of an array of autoantibodies unique to SLE. A positive test for ANAs alone is not diagnostic.

Figure (PageIndex{6}): (a) Systemic lupus erythematosus is characterized by autoimmunity to the individual’s own DNA and/or proteins. (b) This patient is presenting with a butterfly rash, one of the characteristic signs of lupus. (credit a: modification of work by Mikael Häggström; credit b: modification of work by Shrestha D, Dhakal AK, Shiva RK, Shakya A, Shah SC, Shakya H)

Exercise (PageIndex{3})

  1. List the ways antibodies contribute to the pathogenesis of myasthenia gravis.
  2. Explain why rheumatoid arthritis is considered a type III hypersensitivity.
  3. Describe the symptoms of systemic lupus erythematosus and explain why they affect so many different parts of the body.
  4. What is recognized as an antigen in myasthenia gravis?

Table (PageIndex{1}) summarizes the causes, signs, and symptoms of select autoimmune diseases.

Table (PageIndex{1}): Select Autoimmune Diseases
DiseaseCauseSigns and Symptoms
Addison diseaseDestruction of adrenal gland cells by cytotoxic T cellsWeakness, nausea, hypotension, fatigue; adrenal crisis with severe pain in abdomen, lower back, and legs; circulatory system collapse, kidney failure
Celiac diseaseAntibodies to gluten become autoantibodies that target cells of the small intestineSevere diarrhea, abdominal pain, anemia, malnutrition
Diabetes mellitus (type I)Cytotoxic T-cell destruction of the insulin-producing β cells of the pancreasHyperglycemia, extreme increase in thirst and urination, weight loss, extreme fatigue
Graves diseaseAutoantibodies target thyroid-stimulating hormone receptors, resulting in overstimulation of the thyroidHyperthyroidism with rapid and irregular heartbeat, heat intolerance, weight loss, goiter, exophthalmia
Hashimoto thyroiditisThyroid gland is attacked by cytotoxic T cells, lymphocytes, macrophages, and autoantibodiesThyroiditis with goiter, cold intolerance, muscle weakness, painful and stiff joints, depression, memory loss
Multiple sclerosis (MS)Cytotoxic T-cell destruction of the myelin sheath surrounding nerve axons in the central nervous systemVisual disturbances, muscle weakness, impaired coordination and balance, numbness, prickling or “pins and needles” sensations, impaired cognitive function and memory
Myasthenia gravisAutoantibodies directed against acetylcholine receptors within the neuromuscular junctionExtreme muscle weakness eventually leading to fatal respiratory arrest
PsoriasisCytokine activation of keratinocytes causes rapid and excessive epidermal cell turnoverItchy or sore patches of thick, red skin with silvery scales; commonly affects elbows, knees, scalp, back, face, palms, feet
Rheumatoid arthritisAutoantibodies, immune complexes, complement activation, phagocytes, and T cells damage membranes and bone in jointsJoint inflammation, pain and disfigurement, chronic systemic inflammation
Systemic lupus erythematosus (SLE)Autoantibodies directed against nuclear and cytoplasmic molecules form immune complexes that deposit in tissues. Phagocytic cells and complement activation cause tissue damage and inflammationFatigue, fever, joint pain and swelling, hair loss, anemia, clotting, a sunlight-sensitive "butterfly" rash, skin lesions, photosensitivity, decreased kidney function, memory loss, confusion, depression

Key Concepts and Summary

  • Autoimmune diseases result from a breakdown in immunological tolerance. The actual induction event(s) for autoimmune states are largely unknown.
  • Some autoimmune diseases attack specific organs, whereas others are more systemic.
  • Organ-specific autoimmune diseases include celiac disease, Graves disease, Hashimoto thyroiditis, type I diabetes mellitus, and Addison disease.
  • Systemic autoimmune diseases include multiple sclerosis, myasthenia gravis, psoriasis, rheumatoid arthritis, and systemic lupus erythematosus.
  • Treatments for autoimmune diseases generally involve anti-inflammatory and immunosuppressive drugs.
  1. 1 D.A. Van Heel, J. West. “Recent Advances in Coeliac Disease.” Gut 55 no. 7 (2006):1037—1046.
  2. 2 ibid.
  3. 3 P. Martorell et al. “Autoimmunity in Addison’s Disease.” Netherlands Journal of Medicine 60 no. 7 (2002):269—275.
  4. 4 C.C. Mok, C.S. Lau. “Pathogenesis of Systemic Lupus Erythematosus.” Journal of Clinical Pathology 56 no. 7 (2003):481—490.


  • Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University) with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free at

Autoimmune Diseases

A healthy immune system defends the body against disease and infection. But if the immune system malfunctions, it mistakenly attacks healthy cells, tissues, and organs. Called autoimmune disease, these attacks can affect any part of the body, weakening bodily function and even turning life-threatening.

Scientists know about more than 80 autoimmune diseases . Some are well known, such as type 1 diabetes, multiple sclerosis, lupus, and rheumatoid arthritis, while others are rare and difficult to diagnose. With unusual autoimmune diseases, patients may suffer years before getting a proper diagnosis. Most of these diseases have no cure. Some require lifelong treatment to ease symptoms.

Collectively, these diseases affect more than 24 million people in the United States. 1 An additional eight million people have auto-antibodies, blood molecules that indicate a person&rsquos chance of developing autoimmune disease. Autoimmune diseases are affecting more people for reasons unknown. Likewise, the causes of these diseases remain a mystery.

To volunteer for a study seeking causes of and possible treatments for autoimmune diseases, visit this clinical trials website , and you may find one that addresses your condition.

Studies indicate these diseases likely result from interactions between genetic and environmental factors. Gender, race, and ethnicity characteristics are linked to a likelihood of developing an autoimmune disease. 2 Autoimmune diseases are more common when people are in contact with certain environmental exposures, as described below.

Advances in mapping susceptibility variants in the MHC locus

Several computational and empirical challenges complicate the mapping of MHC susceptibility variants. One fundamental challenge is that the MHC has many sequence and structural variations [8], which differ between populations and complicate haplotype inference. Another is that high and extensive LD in the locus makes it difficult to identify causal and independent loci. Non-additive allelic effects in the MHC, and epistatic effects between the MHC and other loci, can also complicate inference of the underlying haplotype structure and disease susceptibility variants.

In recent years, large volumes of sequencing data have made it possible to impute MHC variation on a wide scale, thereby improving our understanding of variability at this locus and of the haplotype structures and enabling reference panels to be created. The availability of accurate reference panels and a large number of genotyped individuals has allowed the identification of independent variants and improved our understanding of their contribution to disease heritability and pathways underlying disease biology [9, 10].

Advances in laboratory-based mapping of MHC variation

Increased throughput, accuracy, and read length in next-generation sequencing (NGS) technologies, as well as the development of user-friendly bioinformatics tools, have enabled higher resolution MHC typing [11]. For instance, whole-genome sequencing (WGS) was successfully used to type HLA-A alleles at full resolution in 1070 healthy Japanese individuals [12] and to fully evaluate HLA-E variability in West African populations [13]. However, the main problem with MHC sequencing using current technologies is the relatively short read lengths, which limit the amount of allelic data that can be generated at a high resolution. Long-range PCR amplification approaches, such as the use of PacBio systems for single molecule real-time sequencing, significantly increase read-length and the resolution for typing MHC alleles [14]. In a comparison of MHC typing in an Indian population using sequence-specific primers, NGS (Roche/454) and single molecule sequencing (PacBio RS II) platforms, higher resolution typing was achieved for MHC class I (HLA-A, HLA-B, and HLA-C ) and class II genes (HLA-DRB1 and HLA-DQB1) using the PacBio platform, with a median read length of 2780 nucleotides [15].

High-density SNP panels, such as the Immunochip platform [7], which has been widely implemented in immunogenetics studies, are a cheaper, faster, and easier alternative to genotyping than direct MHC typing and NGS methods. The Immunochip contains a dense panel of SNPs from the MHC locus, which enables missing classic MHC variants to be inferred in silico, where the imputation is based on the haplotype structure present in large reference panels (Fig. 1). This fine-mapping approach has been used for several autoimmune and inflammatory diseases (Table 1) and for a few infectious diseases (Additional file 1), thereby allowing comprehensive interrogation of the MHC. Moreover, population-specific reference panels made by deep sequencing and used to impute genotypes allow identification of very rare variants and novel single-nucleotide variants in the human genome. This is illustrated by a recent study in which the authors first built a Han Chinese MHC-specific database by deep sequencing the region in 9946 patients with psoriasis and 10,689 healthy controls, and then used this reference panel to impute genotype data to fine-map psoriasis-associated variants [16]. Notably, functional variants in non-coding regions can be identified, as shown in a Japanese cohort of 1070 healthy individuals [12]. These variants would be impossible to discover using SNP microarrays or low coverage sequencing on the same sample size (Fig. 1, Table 1).

Major histocompatibility complex imputation. A reference cohort of subjects for whom both genetic information and classic human leukocyte antigen (HLA) typing is available can be used to infer the missing (untyped) genotypes and amino acids in a discovery cohort. This allows imputed variants to be tested for their associations with a disease of interest. The figure shows imputation points to classic alleles associated with celiac disease risk in the MHC region on chromosome 6. Y tyrosine, S serine, Q glutamine, T threonine, R arginine, E glutamic acid

MHC associations revealed by genome-wide association studies (GWAS) can often not be fine-mapped to a single allele at a single locus rather they comprise independent effects from multiple loci (see “Role of MHC variants in human diseases”). The presence of these multiple, independent effects highlights the heterogeneous nature within and between diseases, which may lead to varying immunological responses. Fine-mapping has also shown that autoimmune diseases share MHC alleles and hence molecular pathways, which are likely to represent targets for shared therapies. For instance, the major associations within MHC class II across autoimmune diseases imply that modulating T-cell receptor (TCR) activation by using peptide-bearing MHC molecules on antigen-presenting cells (APCs) could be therapeutically useful [17]. Shared MHC genetic factors have also been observed between autoimmune and infectious diseases, suggesting that human genetic architecture has evolved in response to natural selection as determined by various infectious pathogens [18].

Advances in computational approaches for mapping MHC variation

Long-range LD between loci and SNP markers across the MHC offers an alternative approach to interrogate functional MHC variation through imputation. The development of different imputation tools using population-specific reference panels has enhanced the interpretation of genotype data derived from genome-wide platforms. MHC imputation is done using reference panels containing both genetic information and classic HLA serotyping, thus allowing identification of MHC allelic and amino acid variants. It is advantageous to impute allele and amino acid variants in the MHC because background sequence diversity causes the binary SNP concept to fail, technically speaking, while many SNPs have more than two alleles and various amino acids can be contained in the same position. For instance, six possible amino acid variants at position 11 in the HLA-DRB1 gene show the strongest association to rheumatoid arthritis (RA) [19]. Two of these (valine and leucine) confer susceptibility to RA, whereas the other four (asparagine, proline, glycine and serine) are protective.

Several tools allowing imputation of classic HLA alleles at four-digit resolution are now available for MHC imputation analysis the most common are SNP2HLA [20], HLA*IMP:01 [21], and an improved HLA*IMP:02 [22]. HLA*IMP:02 outperforms HLA*IMP:01 on heterogeneous European populations and it increases the power and accuracy in cross-European GWAS [22]. Missing data are also better tolerated in HLA*IMP:02, while SNP genotyping platforms must be selected in HLA*IMP:01 [21, 22]. SNP2HLA not only imputes classic alleles but also amino acids by using two European reference panels, one based on data from HapMap-CEPH (90 individuals), and the other on the Type 1 Diabetes Genetics Consortium (T1DGC) study [20]. Another tool, HLA-VBSeq, allows imputation of MHC alleles at full resolution from whole-genome sequence data [23]. HLA-VBSeq does not require prior knowledge of MHC allele frequencies and can therefore be used for samples from genetically diverse populations [23]. It has successfully typed HLA-A alleles at full resolution in a Japanese population and identified rare causal variants implicated in complex human diseases [12].

One commonly used European reference panel for imputation is the T1DGC panel, which covers SNP genotyping and classic HLA serotyping information for 5225 unrelated individuals [20]. Similar population-specific reference panels have been developed for non-European studies to investigate the risk of psoriasis in Chinese populations [16] and of Graves’ disease and RA in Japanese populations. The panels have also been used to impute MHC alleles and amino acids for East Asian and Korean populations [24,25,26].

Using a single reference genome for regions like the MHC, which has substantial sequence and structural diversity, results in poor characterization. To counteract this, an algorithm was developed to infer much of the variation in the MHC it allows genome inference from high-throughput sequencing data using known variation represented in a population reference graph (PRG) [27]. Specifically, the PRG constructed for the MHC combined eight assembled haplotypes, the sequences of known classic HLA alleles, and 87,640 SNP variants from the 1000 Genomes Project [28]. This approach is considered to be an intermediate step between de novo assembly and mapping to a single reference, but requires careful attention to the variation included in the PRG [27].

Despite the development of new tools to investigate MHC variation, the robustness of imputation depends largely on the reference panel and SNP selection. The frequency of alleles can differ between populations, thus highlighting the need to use population-specific reference panels to impute MHC alleles and amino acids. Additionally, the use of many samples is possible for analyzing the non-additive effects of MHC alleles on a wide scale, as described by Lenz et al. for celiac disease (CeD), psoriasis, and type 1 diabetes (T1D) [29]. These non-additive effects could explain our inability to identify susceptibility variants. However, one important limitation of existing imputation methods is that they are limited to the classic MHC alleles and their amino acids. Another limitation is that accuracy is lower for low frequency or rare variants [20, 30] this can be improved by increasing the reference panel size, together with the use of deep sequencing data. Ascertainment bias and lower LD also make it challenging to impute MHC variants in some non-European populations, such as Africans.

MHC genetic variation mediates susceptibility to a wide range of complex diseases, including infectious and autoimmune diseases. The large volume of data generated by recent GWAS has provided an excellent opportunity to apply imputation tools used to fine-map MHC associations to classic alleles and amino acids, as described below for autoimmune diseases. Overall, MHC imputation has proved to be a robust and cost-effective way to identify causal genes underlying disease pathogenesis. Ultimately, knowing the causal genes will help explain disease heritability and lead to a better understanding of the molecular pathways involved in disease pathogenesis. Such work helps to pinpoint potential therapeutic targets.

13.2: Autoimmune Disorders - Biology

ACS Chemical Biology recorded a special podcast, in which Editor-in-Chief Laura Kiessling (Massachusetts Institute of Technology) interviews CRISPR investigator and former Associate Editor Jennifer Doudna (University of California, Berkeley). Listen to the podcast here. A transcript of the interview, which has been lightly edited, is published here as part of our Special Issue on the Chemical Biology of CRISPR.

In Focus
Frontiers in CRISPR

CRISPR-based approaches to genetic engineering are progressing at a rapid pace and present exciting new avenues for science, medicine, and technology. Many of the most cutting-edge advances in genome engineering are encompassed in the Research Articles, Reviews, and Perspectives in this special issue, often with an eye toward future directions for the field. Yet, many questions remain at this new frontier. We asked over 100 CRISPR researchers, including our contributing authors, for their perspectives on some of the most pressing questions surrounding the future of genome engineering and the CRISPR-Cas platform, the challenges that lie ahead, and opportunities for chemists and chemical biologists to drive creative molecular solutions.

Discovering the Genome-Wide Activity of CRISPR-Cas Nucleases

Originally discovered as part of an adaptive bacterial defense system against the invasion of foreign phages, programmable CRISPR-Cas nucleases have emerged as remarkable enzymes with transformative potential for both biological research and clinical application. CRISPR-Cas nucleases likely evolved in their natural context to tolerate imperfect specificity in order to recognize mutant bacteriophages. However, in the context of biological research and clinical applications, high specificity is generally preferred. For therapeutic applications in particular, it is important to carefully and empirically define the genome-wide activity of engineered nucleases, as hundreds of millions to billions of cells may be modified in a single therapeutic dose. Over the past several years, a number of both cell-based and in vitro sensitive and unbiased genome-scale methods to define CRISPR-Cas nuclease specificity have been developed. These methods will play important complementary roles in better understanding their global specificity profiles and identifying optimal nucleases for applications that demand high precision editing. Improving the sensitivity of mutation detection by next-generation sequencing, developing assays to define the functional consequences of unintended off-target activity nuclease activity, and understanding the consequences of individual human genetic variation on gene editing activity will be important areas for future research and development.

Discovery of Oligonucleotide Signaling Mediated by CRISPR-Associated Polymerases Solves Two Puzzles but Leaves an Enigma

The signature component of type III CRISPR-Cas systems is the Cas10 protein that consists of two Palm domains homologous to those of DNA and RNA polymerases and nucleotide cyclases and an HD nuclease domain. However, until very recently, the activity of the Palm domains and their role in CRISPR function have not been experimentally established. Most of the type III CRISPR-Cas systems and some type I systems also encompass proteins containing the CARF (CRISPR-associated Rossmann fold) domain that has been predicted to regulate CRISPR functions via nucleotide binding, but its function in CRISPR-Cas remained obscure. Two independent recent studies show that the Palm domain of Cas10 catalyzes synthesis of oligoadenylates, which bind the CARF domain of the Csm6 protein and activate its RNase domain that cleaves foreign transcripts enabling interference by type III CRISPR-Cas. In one coup, these findings resolved two long-standing puzzles of CRISPR biology and reveal a new regulatory pathway that governs the CRISPR response. However, the full extent of this pathway, and especially the driving forces behind the evolution of this complex mechanism of CRISPR-Cas activation, remains to be uncovered.

The Future of Multiplexed Eukaryotic Genome Engineering
  • David B. Thompson ,
  • Soufiane Aboulhouda ,
  • Eriona Hysolli ,
  • Cory J. Smith ,
  • Stan Wang ,
  • Oscar Castanon , and
  • George M. Church*

Multiplex genome editing is the simultaneous introduction of multiple distinct modifications to a given genome. Though in its infancy, maturation of this field will facilitate powerful new biomedical research approaches and will enable a host of far-reaching biological engineering applications, including new therapeutic modalities and industrial applications, as well as “genome writing” and de-extinction efforts. In this Perspective, we focus on multiplex editing of large eukaryotic genomes. We describe the current state of multiplexed genome editing, the current limits of our ability to multiplex edits, and provide perspective on the many applications that fully realized multiplex editing technologies would enable in higher eukaryotic genomes. We offer a broad look at future directions, covering emergent CRISPR-based technologies, advances in intracellular delivery, and new DNA assembly approaches that may enable future genome editing on a massively multiplexed scale.

Identifying Novel Enhancer Elements with CRISPR-Based Screens
  • Jason C. Klein* ,
  • Wei Chen ,
  • Molly Gasperini , and
  • Jay Shendure*

Enhancers control the spatiotemporal expression of genes and are essential for encoding differentiation and development. Since their discovery more than three decades ago, researchers have largely studied enhancers removed from their genomic context. The recent adaptation of CRISPR/Cas9 to genome editing in higher organisms now allows researchers to perturb and test these elements in their genomic context, through both mutation and epigenetic modulation. In this Perspective, we discuss recent advances in scanning noncoding regions of the genome for enhancer activity using CRISPR-based tools.

Genome Editing: Insights from Chemical Biology to Support Safe and Transformative Therapeutic Applications
  • Renee D. Wegrzyn* ,
  • Andrew H. Lee ,
  • Amy L. Jenkins ,
  • Colby D. Stoddard , and
  • Anne E. Cheever

Programmable nuclease-based genome editing technologies, including the clustered, regularly interspaced, short palindromic repeats (CRISPR)/Cas9 system, are becoming an essential component of many applications ranging from agriculture to medicine. However, fundamental limitations currently prevent the widespread, safe, and practical use of genome editors, especially for human disease interventions. These limitations include off-target effects, a lack of control over editing activity, suboptimal DNA repair outcomes, insufficient target conversion, and inadequate delivery performance. This perspective focuses on the potential for biological chemistry to address these limitations such that newly developed genome editing technologies can enable the broadest range of potential future applications. Equally important will be the development of these powerful technologies within a relevant ethical framework that emphasizes safety and responsible innovation.

Gene Drive: Evolved and Synthetic

Drive is a process of accelerated inheritance from one generation to the next that allows some genes to spread rapidly through populations even if they do not contribute to—or indeed even if they detract from—organismal survival and reproduction. Genetic elements that can spread by drive include gametic and zygotic killers, meiotic drivers, homing endonuclease genes, B chromosomes, and transposable elements. The fact that gene drive can lead to the spread of fitness-reducing traits (including lethality and sterility) makes it an attractive process to consider exploiting to control disease vectors and other pests. There are a number of efforts to develop synthetic gene drive systems, particularly focused on the mosquito-borne diseases that continue to plague us.

Diverse Class 2 CRISPR-Cas Effector Proteins for Genome Engineering Applications

CRISPR-Cas genome editing technologies have revolutionized modern molecular biology by making targeted DNA edits simple and scalable. These technologies are developed by domesticating naturally occurring microbial adaptive immune systems that display wide diversity of functionality for targeted nucleic acid cleavage. Several CRISPR-Cas single effector enzymes have been characterized and engineered for use in mammalian cells. The unique properties of the single effector enzymes can make a critical difference in experimental use or targeting specificity. This review describes known single effector enzymes and discusses their use in genome engineering applications.

Type II-C CRISPR-Cas9 Biology, Mechanism, and Application
  • Aamir Mir ,
  • Alireza Edraki ,
  • Jooyoung Lee , and
  • Erik J. Sontheimer*

Genome editing technologies have been revolutionized by the discovery of prokaryotic RNA-guided defense system called CRISPR-Cas. Cas9, a single effector protein found in type II CRISPR systems, has been at the heart of this genome editing revolution. Nearly half of the Cas9s discovered so far belong to the type II-C subtype but have not been explored extensively. Type II-C CRISPR-Cas systems are the simplest of the type II systems, employing only three Cas proteins. Cas9s are central players in type II-C systems since they function in multiple steps of the CRISPR pathway, including adaptation and interference. Type II-C CRISPR systems are found in bacteria and archaea from very diverse environments, resulting in Cas9s with unique and potentially useful properties. Certain type II-C Cas9s possess unusually long PAMs, function in unique conditions (e.g., elevated temperature), and tend to be smaller in size. Here, we review the biology, mechanism, and applications of the type II-C CRISPR systems with particular emphasis on their Cas9s.

CRISPR Approaches to Small Molecule Target Identification

A long-standing challenge in drug development is the identification of the mechanisms of action of small molecules with therapeutic potential. A number of methods have been developed to address this challenge, each with inherent strengths and limitations. We here provide a brief review of these methods with a focus on chemical-genetic methods that are based on systematically profiling the effects of genetic perturbations on drug sensitivity. In particular, application of these methods to mammalian systems has been facilitated by the recent advent of CRISPR-based approaches, which enable one to readily repress, induce, or delete a given gene and determine the resulting effects on drug sensitivity.

The Promise and Challenge of In Vivo Delivery for Genome Therapeutics

CRISPR-based genome editing technologies are poised to enable countless new therapies to prevent, treat, or cure diseases with a genetic basis. However, the safe and effective delivery of genome editing enzymes represents a substantial challenge that must be tackled to enable the next generation of genetic therapies. In this Review, we summarize recent progress in developing enzymatic tools to combat genetic disease and examine current efforts to deliver these enzymes to the cells in need of correction. Viral vectors already in use for traditional gene therapy are being applied to enable in vivo CRISPR-based therapeutics, as are emerging technologies such as nanoparticle-based delivery of CRISPR components and direct delivery of preassembled RNA–protein complexes. Success in these areas will allow CRISPR-based genome editing therapeutics to reach their full potential.

Editing the Genome Without Double-Stranded DNA Breaks
  • Alexis C. Komor* ,
  • Ahmed H. Badran* , and
  • David R. Liu*

Genome editing methods have commonly relied on the initial introduction of double-stranded DNA breaks (DSBs), resulting in stochastic insertions, deletions, and translocations at the target genomic locus. To achieve gene correction, these methods typically require the introduction of exogenous DNA repair templates and low-efficiency homologous recombination processes. In this review, we describe alternative, mechanistically motivated strategies to perform chemistry on the genome of unmodified cells without introducing DSBs. One such strategy, base editing, uses chemical and biological insights to directly and permanently convert one target base pair to another. Despite its recent introduction, base editing has already enabled a number of new capabilities and applications in the genome editing community. We summarize these advances here and discuss the new possibilities that this method has unveiled, concluding with a brief analysis of future prospects for genome and transcriptome editing without double-stranded DNA cleavage.

Modulating DNA Repair Pathways to Improve Precision Genome Engineering
  • Katherine S. Pawelczak* ,
  • Navnath S. Gavande ,
  • Pamela S. VanderVere-Carozza , and
  • John J. Turchi

Programmable nucleases like the popular CRISPR/Cas9 system allow for precision genome engineering by inducing a site-specific DNA double strand break (DSB) within a genome. The DSB is repaired by endogenous DNA repair pathways, either nonhomologous end joining (NHEJ) or homology directed repair (HDR). The predominant and error-prone NHEJ pathway often results in small nucleotide insertions or deletions that can be used to construct knockout alleles. Alternatively, HDR activity can result in precise modification incorporating exogenous DNA fragments into the cut site. However, genetic recombination in mammalian systems through the HDR pathway is an inefficient process and requires cumbersome laboratory methods to identify the desired accurate insertion events. This is further compromised by the activity of the competing DNA repair pathway, NHEJ, which repairs the majority of nuclease induced DNA DSBs and also is responsible for mutagenic insertion and deletion events at off-target locations throughout the genome. Various methodologies have been developed to increase the efficiency of designer nuclease-based HDR mediated gene editing. Here, we review these advances toward modulating the activities of the two critical DNA repair pathways, HDR and NHEJ, to enhance precision genome engineering.

Repair of a Site-Specific DNA Cleavage: Old-School Lessons for Cas9-Mediated Gene Editing

CRISPR/Cas9-mediated gene editing may involve nonhomologous end-joining to create various insertion/deletions (indels) or may employ homologous recombination to modify precisely the target DNA sequence. Our understanding of these processes has been guided by earlier studies using other site-specific endonucleases, both in model organisms such as budding yeast and in mammalian cells. We briefly review what has been gleaned from such studies using the HO and I-SceI endonucleases and how these findings guide current gene editing strategies.

CRISPRi and CRISPRa Screens in Mammalian Cells for Precision Biology and Medicine

Next-generation DNA sequencing technologies have led to a massive accumulation of genomic and transcriptomic data from patients and healthy individuals. The major challenge ahead is to understand the functional significance of the elements of the human genome and transcriptome, and implications for diagnosis and treatment. Genetic screens in mammalian cells are a powerful approach to systematically elucidating gene function in health and disease states. In particular, recently developed CRISPR/Cas9-based screening approaches have enormous potential to uncover mechanisms and therapeutic strategies for human diseases. The focus of this review is the use of CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) for genetic screens in mammalian cells. We introduce the underlying technology and present different types of CRISPRi/a screens, including those based on cell survival/proliferation, sensitivity to drugs or toxins, fluorescent reporters, and single-cell transcriptomes. Combinatorial screens, in which large numbers of gene pairs are targeted to construct genetic interaction maps, reveal pathway relationships and protein complexes. We compare and contrast CRISPRi and CRISPRa with alternative technologies, including RNA interference (RNAi) and CRISPR nuclease-based screens. Finally, we highlight challenges and opportunities ahead.

Protein Inhibitors of CRISPR-Cas9

Bacteria are under constant predation from viruses, called bacteriophages (phages). This threat has driven the evolution of multiple defense systems, including the CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR associated genes) immune pathway. Phages are not passive bystanders in their CRISPR-mediated demise, however, as many have developed potent protein inhibitors of the bacterial adaptive immune system. Here, I review the work that led to the discovery of many distinct “anti-CRISPR” proteins. Furthermore, I outline how understanding their mechanisms of action has provided a suite of specific and high-affinity reagents to modulate and study CRISPR-Cas applications.

Can CRISPR-Based Gene Drive Be Confined in the Wild? A Question for Molecular and Population Biology

The recent discovery of CRISPR and its application as a gene editing tool has enabled a range of gene drive systems to be engineered with greater ease. In order for the benefits of this technology to be realized, in some circumstances drive systems should be developed that are capable of both spreading into populations to achieve their desired impact and being recalled in the event of unwanted consequences or public disfavor. We review the performance of three broad categories of drive systems at achieving these goals: threshold-dependent drives, homing-based drive and remediation systems, and temporally self-limiting systems such as daisy-chain drives.

Multidimensional Control of Cas9 by Evolved RNA Polymerase-Based Biosensors

Systems to control Cas9 with spatial and temporal precision offer opportunities to decrease side effects, protect sensitive tissues, and create gene therapies that are only activated at defined times and places. Here, we present the design of new Cas9 controllers based on RNA polymerase (RNAP)-based biosensors that produce gRNAs, thereby regulating target knockout. After development and validation of a new abscisic acid-inducible biosensor to control Cas9, we lowered the background of the system using continuous evolution. To showcase the versatility of the approach, we designed biosensors that measure medically relevant protein–protein interactions to drive knockout. Finally, to test whether orthogonal RNAP biosensors could integrate multiple input signals to drive multiple gRNA-based outputs with a single Cas9 protein, we designed an “on-switch/off switch” controller. The addition of one input activates the “on switch” and induces knockout, while the addition of a second input activates the “off switch” and produces a gRNA that directs the Cas9 protein to degrade the “on switch” gRNA vector, thereby deactivating it. This combined activation and deactivation system displayed very low background and inducible target knockout using different combinations of small-molecule treatment. Our results establish engineered RNAP biosensors as deployable Cas9 control elements and open up new opportunities for driving genetic editing technologies by diverse input signals.

Rheostatic Control of Cas9-Mediated DNA Double Strand Break (DSB) Generation and Genome Editing
  • John C. Rose ,
  • Jason J. Stephany ,
  • Cindy T. Wei ,
  • Douglas M. Fowler* , and
  • Dustin J. Maly*

We recently reported two novel tools for precisely controlling and quantifying Cas9 activity: a chemically inducible Cas9 variant (ciCas9) that can be rapidly activated by small molecules and a ddPCR assay for time-resolved measurement of DNA double strand breaks (DSB-ddPCR). Here, we further demonstrate the potential of ciCas9 to function as a tunable rheostat for Cas9 function. We show that a new highly potent and selective small molecule activator paired with a more tightly regulated ciCas9 variant expands the range of accessible Cas9 activity levels. We subsequently demonstrate that ciCas9 activity levels can be dose-dependently tuned with a small molecule activator, facilitating rheostatic time-course experiments. These studies provide the first insight into how Cas9-mediated DSB levels correlate with overall editing efficiency. Thus, we demonstrate that ciCas9 and our DSB-ddPCR assay permit the time-resolved study of Cas9 DSB generation and genome editing kinetics at a wide range of Cas9 activity levels.

A Single-Chain Photoswitchable CRISPR-Cas9 Architecture for Light-Inducible Gene Editing and Transcription
  • Xin X. Zhou ,
  • Xinzhi Zou ,
  • Hokyung K. Chung ,
  • Yuchen Gao ,
  • Yanxia Liu ,
  • Lei S. Qi , and
  • Michael Z. Lin*

Optical control of CRISPR-Cas9-derived proteins would be useful for restricting gene editing or transcriptional regulation to desired times and places. Optical control of Cas9 functions has been achieved with photouncageable unnatural amino acids or by using light-induced protein interactions to reconstitute Cas9-mediated functions from two polypeptides. However, these methods have only been applied to one Cas9 species and have not been used for optical control of different perturbations at two genes. Here, we use photodissociable dimeric fluorescent protein domains to engineer single-chain photoswitchable Cas9 (ps-Cas9) proteins in which the DNA-binding cleft is occluded at baseline and opened upon illumination. This design successfully controlled different species and functional variants of Cas9, mediated transcriptional activation more robustly than previous optogenetic methods, and enabled light-induced transcription of one gene and editing of another in the same cells. Thus, a single-chain photoswitchable architecture provides a general method to control a variety of Cas9-mediated functions.

Generation of Optogenetically Modified Adenovirus Vector for Spatiotemporally Controllable Gene Therapy

Gene therapy is expected to be utilized for the treatment of various diseases. However, the spatiotemporal resolution of current gene therapy technology is not high enough. In this study, we generated a new technology for spatiotemporally controllable gene therapy. We introduced optogenetic and CRISPR/Cas9 techniques into a recombinant adenovirus (Ad) vector, which is widely used in clinical trials and exhibits high gene transfer efficiency, to generate an illumination-dependent spatiotemporally controllable gene regulation system (designated the Opt/Cas-Ad system). We generated an Opt/Cas-Ad system that could regulate a potential tumor suppressor gene, and we examined the effectiveness of this system in cancer treatment using a xenograft tumor model. With the Opt/Cas-Ad system, highly selective tumor treatment could be performed by illuminating the tumor. In addition, Opt/Cas-Ad system-mediated tumor treatment could be stopped simply by turning off the light. We believe that our Opt/Cas-Ad system can enhance both the safety and effectiveness of gene therapy.

Chemical Control of a CRISPR-Cas9 Acetyltransferase
  • Jonathan H. Shrimp ,
  • Carissa Grose ,
  • Stephanie R. T. Widmeyer ,
  • Abigail L. Thorpe ,
  • Ajit Jadhav , and
  • Jordan L. Meier*

Lysine acetyltransferases (KATs) play a critical role in the regulation of transcription and other genomic functions. However, a persistent challenge is the development of assays capable of defining KAT activity directly in living cells. Toward this goal, here we report the application of a previously reported dCas9-p300 fusion as a transcriptional reporter of KAT activity. First, we benchmark the activity of dCas9-p300 relative to other dCas9-based transcriptional activators and demonstrate its compatibility with second generation short guide RNA architectures. Next, we repurpose this technology to rapidly identify small molecule inhibitors of acetylation-dependent gene expression. These studies validate a recently reported p300 inhibitor chemotype and reveal a role for p300s bromodomain in dCas9-p300-mediated transcriptional activation. Comparison with other CRISPR-Cas9 transcriptional activators highlights the inherent ligand tunable nature of dCas9-p300 fusions, suggesting new opportunities for orthogonal gene expression control. Overall, our studies highlight dCas9-p300 as a powerful tool for studying gene expression mechanisms in which acetylation plays a causal role and provide a foundation for future applications requiring spatiotemporal control over acetylation at specific genomic loci.

A Cleavage-Responsive Stem-Loop Hairpin for Assaying Guide RNA Activity
  • Tara R. deBoer ,
  • Noreen Wauford ,
  • Jing-Yi Chung ,
  • Miguel Salvador Torres Perez , and
  • Niren Murthy*

The scope of the CRISPR-Cas9 technology now reaches far beyond genomic engineering. While significant efforts are driving the evolution of this revolutionary biomedical tool, the in vitro cleavage assay remains the standard method implemented to validate the guide RNA that directs endonuclease Cas9 to a desired genomic target. Here, we report the development of an alternative guide RNA validation system called GUIDER. GUIDER features a hairpin loop structure with a proximal guanosine-rich unit, a distal fluorophore unit, and a gRNA-targeting stem component. Cleavage of GUIDER by its complementary RNA-guided Cas9 endonuclease complex yields a fluorescent emission at 525 nm, signaling effective cleavage of the hairpin structure. GUIDER was validated using the model gene target mpcsk9, and it was able to identify the gRNA that could most efficiently cleave the target mpcsk9 gene. The modular design of GUIDER should allow it to have broad applicability in validating gRNAs, and its fluorescent signal output offers a rapid, simple, and quantitative measure of Cas9-mediated DNA cleavage.

CRISPR-Mediated Tagging of Endogenous Proteins with a Luminescent Peptide
  • Marie K. Schwinn* ,
  • Thomas Machleidt ,
  • Kris Zimmerman ,
  • Christopher T. Eggers ,
  • Andrew S. Dixon ,
  • Robin Hurst ,
  • Mary P. Hall ,
  • Lance P. Encell ,
  • Brock F. Binkowski , and
  • Keith V. Wood

Intracellular signaling pathways are mediated by changes in protein abundance and post-translational modifications. A common approach for investigating signaling mechanisms and the effects induced by synthetic compounds is through overexpression of recombinant reporter genes. Genome editing with CRISPR/Cas9 offers a means to better preserve native biology by appending reporters directly onto the endogenous genes. An optimal reporter for this purpose would be small to negligibly influence intracellular processes, be readily linked to the endogenous genes with minimal experimental effort, and be sensitive enough to detect low expressing proteins. HiBiT is a 1.3 kDa peptide (11 amino acids) capable of producing bright and quantitative luminescence through high affinity complementation (KD = 700 pM) with an 18 kDa subunit derived from NanoLuc (LgBiT). Using CRISPR/Cas9, we demonstrate that HiBiT can be rapidly and efficiently integrated into the genome to serve as a reporter tag for endogenous proteins. Without requiring clonal isolation of the edited cells, we were able to quantify changes in abundance of the hypoxia inducible factor 1A (HIF1α) and several of its downstream transcriptional targets in response to various stimuli. In combination with fluorescent antibodies, we further used HiBiT to directly correlate HIF1α levels with the hydroxyproline modification that mediates its degradation. These results demonstrate the ability to efficiently tag endogenous proteins with a small luminescent peptide, allowing sensitive quantitation of the response dynamics in their regulated expression and covalent modifications.

Two-Color 810 nm STED Nanoscopy of Living Cells with Endogenous SNAP-Tagged Fusion Proteins
  • Alexey N. Butkevich ,
  • Haisen Ta ,
  • Michael Ratz ,
  • Stefan Stoldt ,
  • Stefan Jakobs* ,
  • Vladimir N. Belov* , and
  • Stefan W. Hell*

A 810 nm STED nanoscopy setup and an appropriate combination of two fluorescent dyes (Si-rhodamine 680SiR and carbopyronine 610CP) have been developed for near-IR live-cell super-resolution imaging. Vimentin endogenously tagged using the CRISPR/Cas9 approach with the SNAP tag, together with a noncovalent tubulin label, provided reliable and cell-to-cell reproducible dual-color confocal and STED imaging of the cytoskeleton in living cells.

Conformational Dynamics of DNA Binding and Cas3 Recruitment by the CRISPR RNA-Guided Cascade Complex
  • Paul B. G. van Erp ,
  • Angela Patterson ,
  • Ravi Kant ,
  • Luke Berry ,
  • Sarah M. Golden ,
  • Brittney L. Forsman ,
  • Joshua Carter ,
  • Ryan N. Jackson ,
  • Brian Bothner* , and
  • Blake Wiedenheft*

Bacteria and archaea rely on CRISPR (clustered regularly interspaced short palindromic repeats) RNA-guided adaptive immune systems for sequence specific elimination of foreign nucleic acids. In Escherichia coli, short CRISPR-derived RNAs (crRNAs) assemble with Cas (CRISPR-associated) proteins into a 405-kilodalton multisubunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Cascade binds foreign DNA complementary to the crRNA guide and recruits Cas3, a trans-acting nuclease-helicase required for target degradation. Structural models of Cascade have captured static snapshots of the complex in distinct conformational states, but conformational dynamics of the 11-subunit surveillance complex have not been measured. Here, we use hydrogen–deuterium exchange coupled to mass spectrometry (HDX-MS) to map conformational dynamics of Cascade onto the three-dimensional structure. New insights from structural dynamics are used to make functional predictions about the mechanisms of the R-loop coordination and Cas3 recruitment. We test these predictions in vivo and in vitro. Collectively, we show how mapping conformational dynamics onto static 3D-structures adds an additional dimension to the functional understanding of this biological machine.

Called jaundice, it may mean that your immune system is attacking and destroying healthy liver cells. That can lead to a condition called autoimmune hepatitis.


University of Rochester Medical Center, “Disorders of the Immune System.”

Office on Women’s Health, US Department of Health and Human Services, “Autoimmune Diseases Fact Sheet.”

American Academy of Allergy Asthma and Immunology, “Recurrent Infections May Signal Immunodeficiencies.”

National Institute of Arthritis and Musculoskeletal and Skin Diseases, “Autoimmune Diseases.”

CDC: “Hygiene-Related Diseases: Diarrhea,” “Systemic Lupus Erythematosus (SLE),” “Psoriasis.”

National Institute of Neurological Disorders and Stroke, “Vasculitis Syndromes of the Central and Peripheral Nervous Symptoms Fact Sheet.”

Johns Hopkins Bloomberg School of Public Health Pathology, “Autoimmune Disease Research Center: Frequently Asked Questions.”

The Johns Hopkins Lupus Center, “Signs, Symptoms and Co-occuring Conditions.”

University of Washington Orthopaedics and Sports Medicine, “Fatigue.”

National Fibromyalgia and Chronic Pain Association, “Chronic Fatigue Syndrome.”

National Institutes of Health, “Red Itchy Rash? Get the Skinny on Dermatitis.”

World Allergy Organization, “Diagnostic Approach to the Adult With Suspected Immune Deficiency.”

University of Maryland Medical Center, “Photodermatitis.”

University of Pittsburgh Medical Center, “Guillain-Barre Syndrome (GBS.)”

University of Florida Health, “Raynaud’s Phenomenon.”

University of Michigan Health System, “Difficulty Swallowing (Dysphagia.)”

Autoimmune Disease: Meaning, Causes and Treatment

Autoimmunity is a condition characterized by the presence of serum autoantibodies and self-reactive lymphocytes (T-cells).

It manifests sometimes when the body loses immune tolerance (body’s condition to distinguish its own self-antigens from foreign non-self-antigens and not mounting on immunogenic attack against the former) and mounts an abnormal immune attack, either with antibodies or T-cells, against a person’s own self-antigens.

In other words, the immune system of the body, like any complex multi-component system, is subject to failure of some or all of its parts. When the system loses its sense of “self” and begins to attack cells and tissues of the body, the result is autoimmunity. Autoimmune reactions can cause serious damage to cells/tissues and organs and may result in diseases called autoimmune diseases.

Human beings suffer from several autoimmune diseases, some of which are listed in Table 43.2. Some of these diseases are caused by autoantibodies, while others are due to T- cells that cause tissue destruction. Autoimmune diseases arc cither organ-specific or systemic.

In case of organ-specific diseases, the immune response is directed to a target antigen unique to a single gland or organ. As a result, the manifestations are largely limited to that organ the manifestation may be tissue damage or blockage/overstimulation of the normal function of the organ.

Direct cellular damage occurs when lymphocytes or antibodies bind to cell-membrane antigens causing cellular lysis and/or an inflammatory response in the affected organ. Gradually, the damaged cellular structure is replaced by connective tissue leading to a decrease in function of that organ.

Examples of such diseases are autoimmune anaemias insulin- dependent diabetes mellitus, etc. In some diseases, e.g., Grave’s disease, the antibodies bind to hormone receptors and activate them, while in some others, e.g., myasthenia gravis, antibody binding blocks the activation of the receptors.

In case of systemic autoimmune diseases, the immune response is directed towards a broad range of target antigens and, as a result, involves a number of organs and tissues.

These diseases reflect a general defect in immune regulation that results in hyperactive T-cells and B-cells. Tissue damage is widespread, is caused by both T-cells, e.g.. multiple sclerosis and autoantibodies, e.g., systemic lupus erythematous, or by accumulation of immune complexes, e.g., rheumatoid arthritis.

Causes of Autoimmune Disease:

Autoimmune diseases could arise in many possible ways, some of which are as follows:

(1) A tissue antigen that is sequestered from circulation will not be employed in the thymus for clonal deletion of T cells reactive to this antigen. When such an antigen is released into circulation due to trauma to tissues caused by an accident or a viral or bacterial infection, it may induce autoantibody formation.

(2) A number of bacteria and viruses have antigenic determinants that are identical or similar to, normal host cell components. Infection by such pathogens may initiate autoimmunity.

(3) A number of viruses and bacteria can induce nonspecific polyclonal B cells that produce IgM in the absence of TH cells. Such infections could activate B cells reactive to self-antigens and, thereby, cause autoimmunity.

(4) Expression of MHC II in cells that normally do not express them could lead to autoimmunity. Such an inappropriate expression of MHC II is induced by certain agents like IFN-γ.

Treatment of Autoimmune Diseases:

The current therapies provide relief by nonspecific suppression of the immune system. Some agents like cyclosporin A are somewhat selective in that they inhibit only antigen-activated T cells.

More directed approaches are still at experimental level some of these are briefly summarise below:

(i) T cells specific for the concerned antigen are injected into individuals to immunize them against these T cells (T cell vaccination).

(ii) A synthetic peptide differing by only one (or few) amino acid from the auto-antigenic peptide may be used as therapy. The synthetic peptide competes with the auto-antigenic peptide for MHC molecules (peptide blockade of MHC molecules).

(iii) Monoclonal antibodies may be directed against the following:

(a) CD4 (depletes all TH cells),

(b) The α subunit of IL-2 receptor, which is expressed only by antigen-activated TH cells (blocks antigen- activated TH cells),

(c) Specific T cell receptor, and

(d) The specific allelic variant of the MHC molecule that is associated with the autoimmune disease.

(iv) Oral administration of the concerned auto-antigen way induce tolerance to the antigen.

Various mechanisms can alter self components so that they seem foreign to the immune system. New antigenic determinants can be attached to self proteins, or the shape of a self antigen can shift—for a variety of reasons—so that previously unresponsive helper T cells are stimulated and can cooperate with preexisting B cells to secrete autoantibodies. Alteration of the shape of a self protein has been shown to occur in experimental animals and is the most probable explanation for the production of the rheumatoid factors that are characteristic of rheumatoid arthritis. Infectious organisms also can alter self antigens, which may explain why viral infection of specialized cells—such as those in the pancreas that secrete insulin or those in the thyroid gland that make thyroid hormones—often precedes the development of autoantibodies against the cells themselves and against their hormonal products.

Intracellular antigens and antigens found on tissues that are not in contact with the circulation normally are segregated effectively from the immune system. Thus, they may be regarded as foreign if they are released into the circulation as a result of tissue destruction caused by trauma or infection. After sudden damage to the heart, for example, antibodies against heart muscle membranes regularly appear in the blood.

Covid-19: Vaccine candidate may be more than 90% effective, interim results indicate

Could COVID-19 mRNA vaccines cause autoimmune diseases?

Mass vaccination offers the best exit strategy from the COVID-19 pandemic. Pfizer/BioNTech's recent announcement, therefore, is encouraging.[1] Their vaccine candidate was more than 90% effective in preventing COVID-19 infection in participants without prior infection. Being an mRNA vaccine, mass production is cheaper and more straightforward than with other vaccine formulations.

mRNA vaccines effect coded protein production in the recipient’s body. In the case of COVID-19, inert spike (S) antigen proteins are produced. Normally, these enable SARS-CoV-2 coronavirus particles to enter host cells, but therapeutically, inoculation triggers humoral (antibody-mediated) acquired immunity.

Severe/fatal cases of COVID-19 are associated with immune hyperactivation and excessive cytokine release, leading to multiorgan failure. A broad range of mechanisms (with a final common pathway) appear to be involved. However, it has been suggested that molecular mimicry may contribute to this problem, with antibodies to SARS-CoV-2 spike glycoproteins cross-reacting with structurally similar host heptapeptide protein sequences (for example, in interleukin 7 and alveolar surfactant proteins), and raising an acute (auto)immune response against them.[2] Autoinflammatory dysregulation in genetically susceptible individuals, and other autoimmune mechanisms such as epitope spreading and bystander activation, might also contribute to acute but also chronic autoimmunity during and after COVID-19. [3]

In the understandable socioeconomic rush towards mass vaccination without longer-term safety testing, it would seem that an essential stage in any vaccine licensing process should involve careful analysis of the human proteome against vaccine peptide sequences. This should minimize the risks both of acute autoimmune reactions to inoculation and future chronic autoimmune pathology.

1. Mahane E. Covid-19: Vaccine candidate may be more than 90% effective, interim results indicate. BMJ 2020371:m4347.
2. Ehrenfeld M, Tincani A, Andreoli L, et al. Covid-19 and autoimmunity. Autoimmun Rev 202019:102597.
3. Caso F, Costa L, Ruscitti P. Could Sars-coronavirus-2 trigger autoimmune and / or autoinflammatory mechanisms in genetically predisposed subjects? Autoimmun Rev 202019:102524.

Autoimmune Diseases and Their Manifestations on Oral Cavity: Diagnosis and Clinical Management

Oral signs are frequently the first manifestation of autoimmune diseases. For this reason, dentists play an important role in the detection of emerging autoimmune pathologies. Indeed, an early diagnosis can play a decisive role in improving the quality of treatment strategies as well as quality of life. This can be obtained thanks to specific knowledge of oral manifestations of autoimmune diseases. This review is aimed at describing oral presentations, diagnosis, and treatment strategies for systemic lupus erythematosus, Sjögren syndrome, pemphigus vulgaris, mucous membrane pemphigoid, and Behcet disease.

1. Introduction

Increasing evidence is emerging for a steady rise of autoimmune diseases in the last decades [1]. Indeed, the growth in autoimmune diseases equals the surge in allergic and cancer pathology on the other hand, infections are shown to be less frequent in the Western societies [2]. Oral manifestations of autoimmune disease are frequently the primary sign of autoimmune diseases [3]. The dentists can therefore play a pivotal role in the detection and during the following multidisciplinary treatment. Precise and early diagnosis increases the efficiency and efficacy of treatment strategy [4–6]. Therefore, the goal of our review is to present the most common autoimmune diseases that show the first oral clinical signs and symptoms which are a manifestation of the general clinical disease. Our review is presenting details over systemic lupus erythematosus, Sjögren syndrome, pemphigus vulgaris, mucous membrane pemphigoid, and Behcet disease. Every single paragraph reviews the general conditions, and in the second part, we discuss the diagnosis and treatment strategies.

2. Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is a severe and chronic autoimmune inflammatory disease of unknown etiopathogenesis and various clinical presentations. SLE mainly affects women 8 times more likely than men. The worldwide prevalence of SLE ranges between 12 and 50 per 100,000, depending on location and ethnicity [7].

SLE is usually a chronic and progressive disease whose dormancy and progress are fairly regular and in sequence. There are cellular and cell-mediated processes involved in the SLE, even though it has been speculated that the primary involvement is mainly due to cell-mediated immunity and consequential humoral involvement [8]. The immune complex deposits in different organs triggering an inflammatory reaction that leads to organ functional impairment typical of the disease. In the pathogenesis of SLE, the activation of type I IFN pathways, B and T cell dysfunction, and presence of antinuclear antibodies were demonstrated [9]. Anti-DNA antibodies (deoxyribonucleic acid, antinuclear antibodies) are found in the patients’ serum. The proliferation of these antibodies is supported by oestrogens. In some cases, there have been signs of antilymphocyte antibodies. The etiopathogenesis of SLE takes also into account genetic factors [8, 9].

Skin damage is the typical clinical sign of SLE, and it has been recorded in 85% of the cases [8]. Symptoms can vary from simple circular skin lesions to multiorgan impairment, potentially fatal. The most recurrent skin lesion is severe erythema on the surface of the skin exposed to light also, oral discoid lesions are one of the more prevalent presentations of the disease. The so-called malar rash (or butterfly rush) is located on the nose and cheeks, and the erythema is found also on the finger tips. The healing process of these lesions which present a central scar and area around shows recrudescence very often. In SLE, we have involvement of joints, skin, muscles, eyes, lungs, central nervous system, and kidneys. At the joint level, arthralgia and arthritis are frequently associated with the advancement of SLE. The arthralgia has an asymmetric presentation and migratory behaviour [7]. The topography of joint manifestations is very wide. Indeed, it can interest any articular surface mimicking the rheumatoid arthritis. Deformities are generated by the inflammatory process of the tendons rather than degeneration [7]. At the skin level, purpuric manifestations and vitiligo can also be observed [8]. Lesions of retinae, as vasculitis, may injury the nerve fibres causing impairment or loss of vision. Renal disease or lupus nephritis is a grave complication of SLE that affects 30% of patients [10, 11]. The classical clinical manifestation is represented by a regular round or slightly red irregular area. This can be characterized by atrophy or the presence of ulceration. The red area is characterized by typical white radiating striae and telangiectasia. These signs may resemble those of lichen planus, despite the lack of symmetry. Although the oral condition is not major, petechial lesion and gingival bleeding such as desquamative gingivitis, marginal gingivitis, or erosive mucosal lesions have been reported in up to 40% of patients and may indicate serious thrombocytopenia. Many SLE patients may present at the same time Sjögren syndrome [8, 9, 12, 13].

2.1. Diagnosis

SLE diagnosis is based on a multiple-organ condition and the study of antinuclear antibodies at a serum level. The so-called LE cells can be detected in the blood stream. LE cells are mature neutrophils that have swallowed spherical inclusions produced by nuclear components and other cellular elements [8]. Lupus lesions can be confused with erythema multiforme lesions, lichen planus, and vesiculobullous lesions [7]. Moreover, the differential diagnosis has to include lichenoid reactions to dental fillings, traumatic or smoker’s keratosis, and verrucous carcinoma [13]. The demonstration of intact adjacent tissues towards given lesions through histological and immunohistochemical confirmation is still the standard criterion for a definitive diagnosis [8, 12–14].

2.2. Treatment and Prognosis

SLE management is based on prevention, maintenance of states of remission and alleviation of symptoms, and reversal of inflammation [7, 8, 15, 16]. Salicylates and FANS are used in the less severe cases. There are other drugs used, such as hydroxychloroquine (an antimalarial), cortisones, and other immunosuppressants such as azathioprine and cyclophosphamide [8]. High- and medium-potency corticosteroids and calcineurin inhibitors are used as topical therapies for cutaneous manifestation [17]. Protection from sunlight is part of the strategy in order to avoid flare-ups of skin manifestations [7]. The prognosis is often good when the course of the disease is of an intermediate type and only few organs are involved. The disease can also be fatal in the case of kidney conditions with hypertension and rapid evolution towards kidney failure that leads to the patient’s death [8, 18–20].

3. Sjögren Syndrome

Sjögren syndrome is an autoimmune disease affecting salivary and lacrimal glands and causing a reduction of the secretion activity due by lymphocytic infiltration and consequent destruction of the exocrine glands [8]. The lower production of saliva (hyposalivation) causes dryness in the mouth (xerostomia) the deficiency of tears causes xerophthalmia. Although the etiopathology of the Sjögren syndrome is still unknown, humoral- and cell-mediated immunity phenomena are involved in the process as a matter of fact, increased activation of B cells followed by immune complex formation and autoantibody production plays important roles [21]. Genetic and environmental factors can also be part in the pathogenesis of the syndrome [13].

Sjögren syndrome affects 0.5–3% of the entire population and is predominant in women compared to men (9 : 1 ratio). Typically, Sjögren syndrome is detected around 50 years of age. It is important to underline that there are two characteristic surges: just after the menarche and after the menopause [13, 22, 23].

Some patients show clinical signs only confined to the mouth and eyes, while others present a more substantial autoimmune damage. 50% of the cases also have a different autoimmune condition, such as rheumatoid arthritis or systemic lupus erythematosus [8]. Damage to the glands without the evidence of other autoimmune issues is defined as primary Sjögren syndrome. The addition of an autoimmune disease is referred to as secondary Sjögren syndrome [13, 24]. The main signs of the syndrome are related to the oral cavity [8]. The xerostomia is responsible for creating different manifestations of SS at the level of oral cavity. Lack of saliva predisposes patients to develop tooth cavities. The lack of saliva facilitates the accumulation of plaque and their clearance. Edema and inflammations of the gingiva are frequent clinical signs. Moreover, a salivary flow decrease can develop opportunistic infections. Candida is often detected because the lack of lysozyme and immunoglobulins facilitates its development. Radfar et al. and Bayetto and Logan showed an association between Candida and the decreased stimulated salivary flow rate [25, 26]. The Sjögren syndrome affects both major and minor salivary glands. 50% of the cases show an increase in volume, symmetrical on both sides, of the parotid glands. The histological appearance of the hypertrophic glands is characterized by the replacement of the gland tissue by the lymphocytes and the presence of epimyoepithelial islands [24].

In addition to oral symptoms, patients also present irritation and dryness of the eyes, caused by xerophthalmia, as well as by photophobia. Nearly 20% of the patients affected by Sjögren syndrome show signs of the Raynaud phenomenon, a condition that affects fingers and toes [8]. Finally, patients affected by this disease may have arthralgia, myalgia, and asthenia.

The conclusions of different epidemiological studies claim, although newer studies are required to confirm this, that genetical as well as environmental factors play a role in the pathogenesis of the diseases [27, 28]. The syndrome is often accompanied by lab data alteration. 90% of the patients result positive to the rheumatoid factor, an anti-IgG antibody in the patient’s serum. There are also other autoantibodies such as anti-Sjögren A and anti-Sjögren B that can be found in these patients [8].

3.1. Diagnosis

The diagnosis of Sjögren syndrome is basically clinical, supported by oral presentation and laboratory investigations. During recent decades, many classification criteria have been elaborated with the purpose to provide useful guidance for diagnosis by clinicians. The classification made by Shiboski et al. is generally utilized and also endorsed by the American College of Rheumatology [29, 30].

The diagnosis of the syndrome can be confirmed when two out of three of the following conditions are identified: xerostomia, keratoconjunctivitis sicca, and rheumatoid arthritis or another autoimmune disease [8]. Measuring the salivary flow and carrying out a biopsy of the minor salivary glands are two basic diagnostic investigation tests to detect the syndrome [24]. Very often, the xerostomia generates secondary symptoms that can help the clinician to orientate the diagnosis. Indeed, difficulties to speech and metallic sensation in the mouth are characteristic of xerostomia, as well as burning sensation of the oral mucosae [24, 31].

The ophthalmologic test is necessary to detect keratoconjunctivitis sicca. The lacrimal flow is measured by means of special absorbing pads [8]. Damage to the corneas, instead, requires further specific analysis. In most cases, the disease has a chronic and benign progress however, these patients are exposed to a high risk to develop more serious clinical autoimmune issues: lymphoma and Waldenström macroglobulinemia. Periodical check-ups are mandatory in order to control and prevent the risks [8, 32, 33].

3.2. Treatment and Prognosis

The treatment for the Sjögren syndrome is mainly clinical. The use of FANS has a beneficial effect on arthritis. In major cases, corticosteroid and immunosuppressive drugs may be needed. Xerostomia can be regulated by using saliva substitutes such as sprays/gel or by installing an air humidifier. Sugar-free chewing gums may be useful to alleviate the feeling of dryness in the oral cavity, as well as hyperstimulate the salivary production. Methylcellulose artificial tears can alleviate xerophthalmia. Very often, Sjögren syndrome is accompanied by candidiasis produced by Candida albicans. This will require antimycotic treatment [13, 33]. Salivary secretion can be increased by taking pilocarpine. At a dental level, teeth and gums must be protected from the collateral damage caused by xerostomia [8]. Intensive domiciliary as well as professional oral hygiene care is mandatory to avoid complications due to teeth decay or root canal inflammation [34].

4. Pemphigus Vulgaris

Pemphigus vulgaris is a chronic immunomediated disorder. This disease affects the skin and mucosa. Patients affected by pemphigus have immune globulin G autoantibody against desmosomal components like desmoglein-1 and desmoglein-3 [35]. This alters the properties of adhesion cell molecules, producing intraepithelial blisters between the Malpighian epitheliocytes. This phenomenon is called acantolysis of suprabasilar cheratynocites [8, 35].

Although epidemiologically there is no evidence of gender predilection, some studies reported a slight prevalence in women [13]. All ages can be affected, though the highest number of cases is observed in patients in their 40s and 50s [8, 13].

The etiology would seem to be linked to genetic and ethnic factors. The lesions seem to be triggered by different inputs like physical agents, viruses, hormones, drugs, and stress [8, 13].

In over 50% of cases, the first signs of the disease arise in the oral mucosa. Although there is no area predilection, the lesions could be located at the buccal mucosae, soft palate, lower lip, and tongue and, less frequently, at the gingiva [36]. Oral lesions can range from fairly superficial ulcers to small vesicles or blisters. In the oral cavity, the bubbles rapidly break, leaving a painful erosion producing burning sensation [13]. The size of the ulcers is extremely variable. It can be noticed that a detachment of a large area of the surface with the formation of blisters can occur by exerting a slight pressure on the epithelium of these patients. This phenomenon is referred to as the Nikolsky phenomenon [8].

Pemphigus skin lesions are subsequent to oral manifestations. They can arise as simple rashes to erosions, vesicles, blisters, or ulcers. Microscopic examination highlights superficial epithelial damage with the intact basal layer adhering to the basal membranes [8]

4.1. Diagnosis

The pemphigus can be easily confused with other disorders that present lesions like aphthae, lichen planus, candidiasis, and pemphigoid. Often, pemphigus is associated with other autoimmune clinical situations such as the Sjögren syndrome, rheumatoid arthritis, and systemic lupus erythematosus [37, 38]. As a matter of fact, clinical, histopathological, and, in particular, direct and indirect immunofluorescence is mandatory to perform an efficient differential diagnosis. Direct immunofluorescence is performed on the tissue and highlights the local cellular damage (visualization takes place through a special microscope that highlights the fluorescence inside the spinous layer). In the indirect type, the antibodies are detected in the patient’s serum [8, 39].

4.2. Treatment and Prognosis

The pemphigus is a pathology that involves primarily dermatologists although dentists can play an important role in the early diagnosis of the disease as well as in the management of oral manifestations. The treatment involves the administration of high-dose corticosteroids. In addition to these, immunosuppressive drugs such as azathioprine, cyclophosphamide, cyclosporine, and methotrexate are sometimes used. Recently, the use of rituximab has been proposed showing promising results [40, 41]. The titration of circulating antibodies is carried out to evaluate the progress of the disease. In fact, high antibody rates correspond to the most destructive phases of the disease. Their evaluation is also used to check the effectiveness of the treatment [8].

5. Mucous Membrane Pemphigoid

Mucous membrane pemphigoid (MMP) is a group of immune-mediated chronic blistering conditions. The oral mucosa is targeted as well as genital, conjunctival, and skin mucous membranes [8]. The autoantibodies mostly IgA and IgG are located, together with the C3 complement, on the mucosae as well as on epithelial basal membranes [35].

The most affected area is the gingiva, almost 94% of the cases [35], where the pemphigoid lesions give rise to a clinical condition called desquamative gingivitis. It has been said that desquamative gingivitis is not, per se, diagnostic. The lesions show as simple erythema or true ulcerations affecting both the fixed gingiva and the adherent gingiva. Very often, this lesion is confounded with periodontal disease.

However, lesions can also occur in other areas of the oral cavity including the palate, buccal mucosae, lips, tongue, and pharynx.

The symptoms associated with these conditions go from burning sensation and bleeding to masticatory impairment [35]. Pemphigoid blisters are less brittle than those seen in pemphigus and can remain intact in the oral cavity for up to 48 hours [8, 42].

5.1. Diagnosis

The diagnosis of mucous membrane pemphigoid is based on clinical and histological samples. The histologic examination shows the detachment of the epithelium from the underlying connective tissue. Direct immunofluorescence is diriment when there are doubtful histological samples showing a linear involvement at the level of the basal membrane. The immunofluorescence is particularly useful in the differential diagnosis with pemphigus and lichen as well as with periodontal disease and SLE. Epithelial degeneration is not observed the connective tissue appears pervaded by an intense inflammatory infiltrate mainly consisting of plasma cells and eosinophils [8].

5.2. Treatment and Prognosis

The mucous membrane pemphigoid is a chronic disease that requires a continuous treatment strategy although the prognosis is benign. Sometimes, the lesions can only be localized to the gums in other cases, the oral condition is wider. In less severe cases, the lesions can be treated by topical corticosteroid gel application although in some selected cases, it is coupled with dapsone (diaminodiphenyl sulfone). In the most severe forms, the treatment must be carried out systemically. Often, the pathology can be difficult to resolve, tending to respond rather late to therapy. It is crucial to monitor the presence of eye lesions to prevent ocular damage, such as injury to the cornea, conjunctiva, or eyelids [13, 35].

6. Behcet Disease

Behcet syndrome is an autoimmune, multisystemic disease of unknown etiology. It is typically characterized by at least two of the three key typical factors: oral ulcers, genital ulcers, and eye inflammation. Although its original definition is linked to dermatologic pathology, Behcet disease is often characterized by neurological and vascular involvement. It usually affects individuals in their 30s and shows no evidence of gender predilection. The greatest incidence of the disease is observed in Mediterranean and Asian populations with a marked prevalence in Turkey. The demonstration of an autoimmune genesis is given by the presence of antimucous autoantibodies, together with the association of the disease with the HLA configurations B5 and B51 [8, 43].

The mucocutaneous lesions are very often the first sign of the presence of Behcet syndrome. Their recognition is a key factor for early diagnosis, and they permit a more favourable prognosis [44]. The oral lesions are ulcers of the oral mucosae indistinguishable from the conventional aphthae of the oral mucosa. They are painful and characterized by cyclic presentation. They are localized at the lips, buccal mucosa, soft palate, and tongue. At the beginning, the lesion shows as an erythematous lesion, followed by an evolution in ulcers. Their dimensions can vary from few millimeters to centimeters [8, 44].

The genital ulcers are smaller and are located at the level of the scrotum, on the base of the penis, or on the labia majora.

Ocular lesions are present in 30–70% of the cases [43]. They show up as an initial form of photophobia, followed by uveitis and conjunctivitis. In some cases, they were found to be associated with glaucoma and cataract [43].

The skin lesions have a papular or pustular appearance and are mainly localized to the trunk or limbs.

6.1. Diagnosis

It has been said that there are not pathognomonic laboratory findings [43]. In order to diagnose the Behcet syndrome, according to the ISG criteria [45], at least two of the main features (oral, genital, or ocular lesions) must be present when another clinical explanation is excluded. Indeed, the differential diagnosis is a challenge considering that oral aphthous lesions are very common in the general population. Moreover, aphthous lesions are linked to HIV, Crohn’s disease, sarcoidosis, and SLE, given that the dual-site-specific ulcerations seem to be the unique sign used to differentiate the Behcet syndrome from different pathologies cited above [43].

6.2. Treatment and Prognosis

The treatment of Behcet syndrome is based on the use of local and systemic cortisones per se or coupled with immunosuppressant drugs. The use of immunosuppressive drugs is justified by the lack of prevention of relapses due to the monocorticosteroid treatment strategy [43]. The main objective of Behcet syndrome patient care is to treat in time the oral mucocutaneous lesions in order to hinder the progression of the disease and to prevent the irreversible organ involvement in particular during the active phase [44]. Behcet syndrome could be fatal especially in the case of vascular involvement: aneurism rupture and thrombosis are the main causes of death.

Conflicts of Interest

The authors declare that there are no conflicts of interest regarding the publication of this paper.


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