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In this video I will show panel repair bypass method.LCD LED panel repair without bonding machine.This video is for experiment purpose only for beginners.Pan...

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Humans and other mammals live in a world that is heavily populated by both pathogenic and non-pathogenic microbes, and that contains a vast array of toxic or allergenic substances that threaten normal homeostasis. The community of microbes includes both obligate pathogens, and beneficial, commensal organisms, which the host must tolerate and hold in check in order to support normal tissue and organ function. Pathogenic microbes possess a diverse collection of mechanisms by which they replicate, spread and threaten normal host functions. At the same time that the immune system is eliminating pathological microbes and toxic or allergenic proteins, it must avoid responses that produce excessive damage of self-tissues or that might eliminate beneficial, commensal microbes. Our environment contains a huge range of pathogenic microbes and toxic substances that challenge the host by a very broad selection of pathogenic mechanisms. It is not surprising, therefore, that the immune system uses a complex array of protective mechanisms to control and usually eliminate these organisms and toxins. A general feature of the immune system is that these mechanisms rely on detecting structural features of the pathogen or toxin that mark it as distinct from host cells. Such host-pathogen or host-toxin discrimination is essential to permit the host to eliminate the threat without damaging its own tissues.

The mechanisms permitting recognition of microbial, toxic, or allergenic structures can be broken down into two general categories: i) hard-wired responses that are encoded by genes in the host’s germ line and that recognize molecular patterns shared both by many microbes and toxins that are not present in the mammalian host; and ii) responses that are encoded by gene elements that somatically rearrange to assemble antigen-binding molecules with exquisite specificity for individual unique foreign structures. The first set of responses constitutes the innate immune response. Because the recognition molecules used by the innate system are expressed broadly on a large number of cells, this system is poised to act rapidly after an invading pathogen or toxin is encountered and thus constitutes the initial host response. The second set of responses constitutes the adaptive immune response. Because the adaptive system is composed of small numbers of cells with specificity for any individual pathogen, toxin or allergen, the responding cells must proliferate after encountering the antigen in order to attain sufficient numbers to mount an effective response against the microbe or the toxin. Thus, the adaptive response generally expresses itself temporally after the innate response in host defense. A key feature of the adaptive response is that it produces long-lived cells that persist in an apparently dormant state, but that can re-express effector functions rapidly after another encounter with their specific antigen. This provides the adaptive response with the ability to manifest immune memory, permitting it to contribute prominently to a more effective host response against specific pathogens or toxins when they are encountered a second time, even decades after the initial sensitizing encounter.

Broadly defined, the innate immune system includes all aspects of the host’s immune defense mechanisms that are encoded in their mature functional forms by the germ-line genes of the host. These include physical barriers, such as epithelial cell layers that express tight cell-cell contacts (tight junctions, cadherin-mediated cell interactions, and others), the secreted mucus layer that overlays the epithelium in the respiratory, gastrointestinal and genitourinary tracts, and the epithelial cilia that sweep away this mucus layer permitting it to be constantly refreshed after it has been contaminated with inhaled or ingested particles. The innate response also includes soluble proteins and bioactive small molecules that are either constitutively present in biological fluids (such as the complement proteins, defensins, and ficolins–

Unlike the innate mechanisms of host defense, the adaptive immune system manifests exquisite specificity for its target antigens. Adaptive responses are based primarily on the antigen-specific receptors expressed on the surfaces of T- and B-lymphocytes. Unlike the germ-line-encoded recognition molecules of the innate immune response, the antigen-specific receptors of the adaptive response are encoded by genes that are assembled by somatic rearrangement of germ-line gene elements to form intact T cell receptor (TCR) and immunoglobulin (B cell antigen receptor; Ig) genes. The assembly of antigen receptors from a collection of a few hundred germ-line-encoded gene elements permits the formation of millions of different antigen receptors, each with potentially unique specificity for a different antigen. The mechanisms governing the assembly of these B and T cell antigen receptors and assuring the selection of a properly functioning repertoire of receptor-bearing cells from the huge randomly generated potential repertoire will be introduced below and discussed in more detail in chapters 3 and 4.

An intact immune response includes contributions from many subsets of leukocytes. The different leukocyte subsets can be discriminated morphologically by a combination of conventional histological stains, and by analysis of the spectrum of glycoprotein differentiation antigens that are displayed on their cell membranes. These differentiation antigens are detected by their binding of specific monoclonal antibodies. These cell phenotype-determining antigens are assigned cluster of differentiation (CD) numbers. There are currently over 350 defined CD antigens. Updates are issued by Human Cell Differentiation Molecules (HCDM), an organization that organizes periodic Human Leukocyte Differentiation Antigen (HLDA) workshops at which newly identified cell surface molecules are defined and registered. The next HLDA workshop (HLDA9) will be held in Barcelona, Spain, and the summary of authorized CD molecules will be published at http://www.hcdm.org/.

Myeloid stem cells (also termed common myeloid progenitors) give rise to the several different forms of granulocytes, to megakaryocytes and platelets, and to erythrocytes. Cells of the granulocyte lineage that play prominent immune functions include neutrophils, monocytes, macrophages, eosinophils, basophils, and mast cells. In some mammals, platelets also release immunologically significant mediators that expand their repertoire beyond their role in hemostasis. The immune functions of the classical granulocytes have been inferred from the immunologically active molecules they produce and from their accumulation in specific pathological conditions. For example, neutrophils produce large quantities of reactive oxygen species that are cytotoxic to bacterial pathogens. They also produce enzymes that appear to participate in tissue remodeling and repair following injury. Neutrophils accumulate in large quantities at sites of bacterial infection and tissue injury and possess prominent phagocytic capabilities that permit them to sequester microbes and particulate antigens internally where they can be destroyed and degraded. Thus, it is clear that they play a major role in clearance of microbial pathogens and repair of tissue injury.

Like neutrophils, monocytes and macrophages are also highly phagocytic for microbes and particles that have been marked for clearance by binding Ig and/or complement. They appear to be mobilized shortly after the recruitment of neutrophils and they persist for long periods at sites of chronic inflammation and infection. In addition to participating in acute inflammatory responses, they are prominent in granulomatous processes throughout the body. They use production of nitric oxide as a major mechanism for killing microbial pathogens, and also produce large amounts of cytokines such as IL-12 and interferon (IFN)-γ giving them a regulatory role in adaptive immune responses. Depending on the nature of activating signals that are present when macrophages differentiate from immature precursor cells and when they receive their first activation signal, macrophages can adopt one of several phenotypes.

A major challenge faced by the immune system is to identify host cells that have been infected by microbes that then use the cell to multiply within the host. Simply recognizing and neutralizing the microbe in its extracellular form does not effectively contain this type of infection. The infected cell that serves as a factory for production of progeny microbes must be identified and destroyed. In fact, if the immune system were equally able to recognize extracellular microbes and microbially infected cells, a microbe that managed to generate large amounts of extracellular organisms or antigen might overwhelm the recognition capacity of the immune system, allowing the infected cells to avoid immune recognition. A major role of the T cell arm of the immune response is to identify and destroy infected cells. T cells can also recognize peptide fragments of antigens that have been taken up by APC through the process of phagocytosis or pinocytosis. The way the immune system has evolved to permit T cells to recognize infected host cells is to require that the T cell recognize both a self-component and a microbial structure. The elegant solution to the problem of recognizing both a self-structure and a microbial determinant is the family of MHC molecules. MHC molecules (also called the human leukocyte-associated [HLA] antigens) are cell surface glycoproteins that bind peptide fragments of proteins that either have been synthesized within the cell (class I MHC molecules) or that have been ingested by the cell and proteolytically processed (class II MHC molecules).

Molecular models derived from crystal structures of class I (A–C) and class II (D–F) HLA molecules. A, the class I α1, α2, and α3 domains are shown (light blue) in non-covalent association with the β2m molecule. Coils represent α-helices, and broad arrows represent β-strands. Anti-parallel β-strands interact to form β-sheets. The α-helices in the α1 and α2 domains form the sides and floor of a groove that binds processed antigenic peptides (yellow). The transmembrane and intracytoplasmic portions of the heavy chain are not shown. B, top view of the α1 and α2 domains displaying the antigenic peptide in a molecular complex for recognition by the TCR of a CD8+ T cell (recognition site outlined by pink rectangle). C, side view of the α1 and α2 domains highlighting the TCR contact points on both the α-helices and antigenic peptide. D, side view of the HLA class II molecule showing the α chain (light blue) and the β chain (dark blue). In the class II protein, the peptide-binding groove is made of α helices in both the α1 and β1 domains and a β-sheet formed again by both the α1 and β1 domains. E, top view of the both the α1 and β1 domains and the processed antigenic peptide fragment as they would be seen by the TCR of a CD4+ T cell. F, side view highlighting the α1 and β1 domains and the antigenic peptide. Modified with permission from Bjorkman.

A key biological consequence of requiring the T cell to recognize antigenic peptides only when they are bound in the groove of an HLA molecule is that this permits the T cell to ignore free extracellular antigen, and to focus rather on cells that contain the antigen. In the case of cells that are infected by a pathogenic microbe, this permits the T cells to focus their response on the infected cells. The α3 domain of the class I heavy chain interacts with the CD8 molecule on cytolytic T cells. This restricts recognition of antigenic peptides that are presented in class I HLA molecules to CD8+ cytolytic T cells. The binding of CD8 expressed by the T cell to the α3 domain of the class I molecule expressed by the APC strengthens the interaction of the T cell with the APC and helps assure that full activation of the T cell occurs.

Generally, the antigenic peptides that are found bound in the peptide binding groove of the HLA class I molecules are derived from proteins synthesized within the cell bearing the class I molecules. They are, consequently, described as ‘endogenous’ antigens. The molecular machinery that generates peptide fragments from intracellular proteins and directs them into the grooves of the class I molecules is increasingly well understood (Figure 4). Peptide fragments are generated from cellular proteins by the action of the proteasome, a proteolytic factory composed of over 25 subunits.Figure 2). The LMP2 and LMP7 proteins alter the proteolytic specificity of the proteasome, enhancing the production of peptide fragments of appropriate length and charge for binding in the groove of the HLA class I proteins. The addition of another IFN-γ induced protein termed the PA28 proteasome activator also enhances the generation of antigenic peptides that are favorable for presentation in HLA class I molecules.Figure 2). Once in the ER, the peptides are loaded into the class I protein binding groove under the direction of the ER protein tapasin with the help of the calcium-binding chaperone protein calreticulin and the oxidoreductase Erp57.,2-microglobulin, the class I protein is maintained in a conformation that favors interaction with peptide fragments by association with the chaperone protein calnexin. Interaction with β2-microglobulin stabilizes the complex, causing dissociation of calnexin, and permitting transport of the peptide-loaded class I molecule via the Golgi complex into exocytic vesicles that release the intact complexes onto the cell surface. This pathway is well-adapted to delivering viral peptides produced in a virus infected cell to the cell surface bound to class I HLA molecules in a form that can be recognized by cytotoxic CD8+ T cells. It may also be used to present tumor specific protein fragments that may be useful targets for anti-tumor immunotherapy.

Like the class I molecules, the class II HLA molecules consist of two polypeptide chains, but in this case both are MHC-encoded transmembrane proteins and are designated α and β. There are three major class II proteins designated HLA-DR, HLA-DQ, and HLA-DP.Figure 2). Pairing of the common a chain with the DRB1 chain produces the HLA-DRB1 protein. Over 500 HLA-DRB1 alleles have been defined. Pairing of the common α chain with the DRB3 chain produces molecules designated HLA-DRB2 through HLA-DRB9. There are in total 60 HLA-DRB2 through HLA-DRB9 alleles. The HLA-DQ sub-region encodes 1 polymorphic α chain (25 alleles) and 1 polymorphic β chain (72 alleles). The HLA-DP sub-region encodes 1 polymorphic α chain (16 alleles) and 1 polymorphic β chain (118 alleles). Because both the α chains and the β chains of the HLA-DQ and HLA-DP proteins are polymorphic, each person can express 4 different HLA-DQ and 4 different HLA-DP proteins based on pairing between the gene products of both the maternal and the paternal chromosome. Furthermore, because the minimally polymorphic HLA-DR α chain can pair with an HLA-DRB1 and an HLA-DRB3 chain from both the maternal and the paternal chromosome, each person can express 4 distinct HLA-DR proteins as well. Each of these has the potential to bind a large repertoire of antigenic peptides, making it difficult for a pathogenic microbe to mutate its structure to a form that cannot be recognized by binding in an HLA class II protein.

Antigens that are presented by class II proteins are loaded into the class II peptide-binding groove via the ‘exogenous’ pathway that starts by endocytosis or phagocytosis of extracellular proteins (Figure 5). The exogenous antigens include antigenic proteins of extracellular pathogens such as most bacteria, parasites, and virus particles that have been released from infected cells and taken up by phagocytosis as well as environmental proteins and glycoproteins such as pollens and venoms, and alloantigens. The ingested antigens are processed to linear peptide fragments by proteolysis after fusion of lysosomes with the phagocytic vacuoles or endosomes to form an acidic compartment.+ endosome to the plasma membrane.

In the endoplasmic reticulum (ER), newly synthesized class II proteins associate with the help of calnexin with an invariant chain protein that protects the antigen-binding groove of the class II molecule until it is transported to the class II+ endosomal protein loading compartment. Exogenous antigens are taken up by phagocytosis or endocytosis, digested by the action of lysosomal enzymes, and transported to the class II+ peptide loading compartment for loading into a class II protein. There the invariant chain is proteolytically degraded and replaced by antigenic peptide with the help of the HLA-DM protein. The assembled class II protein-peptide complex is then delivered to the plasma membrane for recognition by CD4+ T cells. Modified with permission from Huston.

Antigen presentation by class I and class II HLA molecules to CD8+ and CD4+ T lymphocytes is limited to protein antigens. Initially, it was thought that responses to polysaccharide antigens and lipid antigens was restricted to T cell-independent responses that resulted in direct activation of B cells by an antigen with a repeating structure; however, recently it has become clear that there is a class of T cells that recognizes antigens presented by molecules that are not classical HLA class I or class II antigens. One of these classes of T cells uses an antigen receptor composed of α and β chains and recognizes lipid antigens that are presented bound to CD1 molecules.2-microglobulin. There are 5 human CD1 isoforms designated CD1a-CD1e, encoded by linked genes that are not associated with the MHC. X-ray crystallography shows that the α1 and α2 domains of CD1 molecules associate like class I MHC molecules to form a binding groove that can accommodate glycolipid components of microbial pathogens.Figure 2). They share structural characteristics with the class I protein heavy chains but appear not to associate with β2microglobulin and not to bind antigenic peptides. Rather, they act as stress-induced molecules that are targets for intestinal γδ T cells, further expanding the repertoire of molecules that can contribute to activation of responding T lymphocytes. In addition to the two functional MICA and MICB genes, there are at least three inactive MIC pseudogenes encoded within the class I region of the MHC (Figure 2).

Each individual T cell bears antigen receptors of a single specificity. A repertoire of T cells that can protect against the vast universe of microbial pathogens must, therefore, include a very large number of cells encoding a huge array of discrete TCR. These receptors are somatically assembled from variable, diversity, and joining gene elements to generate mature VαJα chains and VβDβJβ chains (see chapter 3). The assembly of these gene elements is initiated by the lymphoid-specific RAG1 and RAG2 proteins which cleave the DNA near the V, D, and J segments and the gene segments are rejoined by a collection of non-lymphoid-specific DNA repair enzymes including DNA-dependent protein kinase (DNA-PK), Ku, XRCC4, XLF, DNA ligase IV, and the Artemis nuclease.Figure 6), a complex lymphoid organ located in the anterior mediastinum at the base of the neck.AIRE (autoimmune regulator). Defective expression of AIRE gives rise to the severe autoimmune syndrome called autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED).

A, the complete TCR complex includes the rearranged TCR α and β chains and also the CD3γ, CD3δ, CD3ε, and CD3ζ chains. The CD3 chains contain ITAMs in their cytoplasmic domains that can be phosphorylated to activate the intracellular signaling cascade for T cell activation. The signaling protein tyrosine kinases Lck and Fyn associate with the intracellular portions of the CD4 and CD3 chains respectively. TCR engagement by MHC plus peptide without the presence of costimulatory proteins fails to activate phosphorylation of the CD3 ITAMs and results in anergy. B, TCR engagement by MHC plus peptide with costimulatory interactions between CD28 on the T cell and CD80 or CD86 (B7.1 or B7.2) on the APC results in Lck- and Fyn-dependent phosphorylation of the CD3 chains, and recruitment of the adapter protein ZAP-70 to the CD3 complex. This leads to phosphorylation of ZAP-70, which induces the downstream program of T cell activation. C, polyclonal activation of T cells can be elicited by superantigens which interact outside the peptide binding groove with the β1 chain of the class II molecule and with all Vβ chains of a particular subclass. This activates CD4-independent, but Fyn-dependent phosphorylation of the CD3 chains, recruitment and phosphorylation of ZAP-70, and cell activation.

During their progress through the thymus, αβ T cells differentiate into discrete subpopulations, each with defined repertoires of effector functions. The major subsets are defined by their selective surface expression of CD4 or CD8. In the thymus, most developing T cells follow a developmental program in which in the cortex they first express neither CD4 nor CD8 (double negative) and then express both CD4 and CD8 (double positive [DP]).−CD8+, and those that are selected on class II MHC molecules become CD4+CD8−. The fact that the CD4 molecule contributes to a stable interaction of the developing T cell with class II MHC molecules on the selecting APC and that CD8 contributes to interactions with class I molecules is central to the association of CD4 with class II MHC restricted antigen recognition and of CD8 with class I restricted antigen recognition. Cells that survive positive selection then move to the thymic medulla for negative selection and export to the periphery. In the blood and secondary lymphoid organs, 60–70% of T cells are CD4+CD8− (CD4+) and 30–40% are CD4−CD8+ (CD8+). CD4+ T cells are generally designated ‘helper cells’ and activate both humoral immune responses (B cell help) and cellular responses (delayed type hypersensitivity responses, others). CD8+ cells show a major cytotoxic activity against cells infected with intracellular microbes and against tumor cells, but also contain regulatory cells that down-regulate immune responses (suppressor cells). A portion of the circulating CD4+ T cells play an important regulatory role that acts to down modulate immune responses. These regulatory T (Treg) cells fall into two groups. The first group develops its regulatory function in the thymus and is known as natural Treg cells. These cells are characterized by surface expression of the CD4 and CD25 antigens and by nuclear expression of the forkhead box P3 transcription factor (Foxp3) that is essential for their development. A major portion of this population’s regulatory activity is due to its secretion of the immunomodulatory cytokines TGFβ and IL-10.reg cells requires cell-cell contact. In this situation, it has been reported that TGFβ acts in a membrane-associated form.reg cells is thought to differentiate in the periphery from naïve CD4+ T cells. Because they appear to develop in response to stimulation with specific antigen, they are called adaptive or induced Treg cells. Their differentiation appears to depend on the presence of IL-10 during their initial activation. Expression of Foxp3 is variable in this subset, and IL-10 is a prominent secreted product with TGFβ also participating.reg phenotype.

Conventional antigens bind to a subset of MHC molecules and to a very small fraction of the huge array of TCR. Thus, a conventional peptide antigen activates only a very small fraction of the total pool of T cells. Superantigens, in contrast, are microbial products that bind to large subsets of TCR proteins and MHC molecules, so that a single superantigen can activate up to 20% or more of the total T cells in the body. The superantigen does this by binding without proteolytic processing to the MHC molecule outside of the antigen-binding groove and to TCR proteins outside of their antigen-MHC binding site (Figure 7). For example the Toxic Shock Syndrome Toxin-1 (TSST-1) produced by Staphylococcus aureus can activate all T cells with TCR that use the Vβ2 and Vβ5.1 chains. The activation of large numbers of T cells induced by superantigens results in the massive release of cytokines producing clinical conditions such as toxic shock syndrome.

B cells constitute approximately 15% of peripheral blood leukocytes. They are defined by their production of Ig. Except as noted below, Ig molecules are composed of two identical 50 kDa heavy chains and two identical 25 kDa κ or λ light chains (see chapter 3). The amino terminal portions of the heavy and light chains vary in amino acid sequence from one antibody molecule to another. These variable portions are designated VH and Vκ or Vλ, respectively. The juxtaposition of one VH segment and one Vκ or Vλ segment creates the antigen-binding portion of the intact Ig molecule. The variable regions of both the heavy and light chains contain three sub-regions that are highly variable between different antibody molecules. These hypervariable sequences are brought together in the Ig protein to form the antigen-binding domain of the molecule. Thus, each Ig has two identical antigen binding sites. The carboxyl terminal portions of the heavy and light chains are constant in each subclass of antibody. The heavy chain constant regions pair to form the Fc domain of the molecule that is responsible for most of the effector functions of the Ig molecule, including binding to Fc receptors and activating the complement system.

B cells differentiate from hematopoietic stem cells in the bone marrow. It is here that their antigen receptors (surface Ig) are assembled from genetic building blocks in a RAG1/RAG2-mediated process similar to that used for the production of functional TCR.H), diversity (DH), and joining (JH) region. Joining of genes encoding variable and constant light chain gene elements generates the amino terminal portion of the light chain. The VDJ junctions formed by this recombination make up the 3rd hypervariable region that contributes to the antigen-binding site. The amino acid sequence diversity of the 3rd hypervariable region is the result of combinatorial V-D-J joining, and also of non-gene-encoded sequences added into the junction sites by the action of the enzyme TdT that is expressed in developing B cells during the time this gene rearrangement is occurring.

Differentiation of stem cells to the B lineage depends on bone marrow stromal cells that produce IL-7. The developing B cells follow a program of differential surface antigen expression and sequential heavy and light chain gene rearrangement (Figure 8). First, the recombinase enzyme complex catalyzes the fusion of one of the DH region genes to a JH region gene with the deletion of the intervening DNA sequences. This DHJH recombination occurs on both chromosomes. Next, the recombinase joins one of the VH region genes to the rearranged DHJJ gene. TdT is expressed during this period, resulting in the addition of random nucleotides into the sites of DH-J and VH-DHJH joining, adding to the potential diversity of amino acid sequences encoded by the rearranged VHDHJH gene. The rearranged VHDHJH element forms the most 5’ exon of this rearranged heavy chain gene, and is followed downstream by exons encoding the constant region of the m chain that pairs with a light chain to produce IgM and farther downstream by exons encoding the constant region of the d chain that is used to make IgD. μ chains and δ chains are produced as a result of alternative RNA splicing of the VHDHJH exon to either the μ exons or the δ exons. If the rearrangements of the VH, DH, and JH elements yields a heavy chain transcript that is in-frame and encodes a functional heavy chain proteins, this heavy chain is synthesized and pairs in the cell with two proteins, λ5 and VpreB, which act as a surrogate light chain (Figure 8). Expression of this pre-B cell receptor on the cell surface prevents VH to DHJH rearrangement on the other chromosome, assuring that the developing B cell produces only one antigenic specificity. This process is called allelic exclusion. If the first VHDHJH rearrangement is out of frame and does not produce a functional heavy chain protein, then a VH gene proceeds to rearrange on the other chromosome in a second attempt to generate a successful heavy chain rearrangement. If this second rearrangement is unsuccessful, the cell undergoes apoptosis and is removed.

Naïve B cells express on their cell surfaces IgM and IgD. These two Ig isotypes are expressed by alternative splicing of the same VHDHJH exon to the m and d heavy chain exons. For all heavy chain genes, alternative splicing also permits expression of both membrane-bound (splicing in a transmembrane exon) and secreted (transmembrane exon spliced out) antibody. As B cells mature under the influence of helper T cells, T cell-derived cytokines induce isotype switching. Isotype switching is a process of DNA rearrangement mediated in part by the RNA-editing enzyme activation-induced cytidine deaminase (AID), uracil-DNA glycosylase (UNG), the endonuclease APE1, and the DNA repair enzyme DNA-PK. Switching moves the rearranged VHDHJH exon into a position immediately upstream of alternative heavy chain exons. This permits a functionally rearranged VHDHJH exon to be used to produce antibodies of different isotypes but the same antigenic specificity.

At the same time as B cells undergo isotype switching, an active process produces mutations, apparently randomly, in the antigen-binding portions of the heavy and light chains. This process, designated somatic mutation, also appears to require AID, UNG, APE1, and DNA repair enzymes.

Isotype switching and somatic mutations are strongly associated with the development of B cell memory. Memory responses, defined as rapid induction of high levels of high affinity antibody after secondary antigen challenge, are characterized by production of IgG, IgA, and IgE antibodies, and by somatic mutations in the antigen-binding domains of the heavy and light chains of these antibodies.

B cells can also be activated successfully without T cell help. T cell independent B cell activation occurs without the assistance of T cell co-stimulatory proteins. In the absence of co-stimulators, monomeric antigens are unable to activate B cells. Polymeric antigens with a repeating structure, in contrast, are able to activate B cells, probably because they can cross-link and cluster Ig molecules on the B cell surface. T cell independent antigens include bacterial lipopolysaccharide (LPS), certain other polymeric polysaccharides, and certain polymeric proteins. Somatic mutation does not occur in most T cell independent antibody responses. Consequently, immune memory to T cell independent antigens is generally weak. This is why it is difficult to create fully protective vaccines directed against polysaccharide components of microbes. Covalent attachment of the polysaccharide component to a carrier protein, in order to recruit T cell help to the response, can induce a beneficial memory response. The value of coupling a polysaccharide antigen to a carrier protein was observed in the Haemophilus influenzae type B vaccine. The original polysaccharide vaccine provided low antibody titers, and no protection for children less than 18 months of age. The current conjugate vaccine generally provides protection beginning at 2–4 months of age.

Cytokines act on cells via transmembrane cell surface receptors. Binding of the cytokine to the receptor elicits its cellular response by activating an intracellular signal transduction pathway that ultimately leads to induction of new gene transcription and synthesis of new cellular proteins. Most cytokine receptors signal using one of the Janus kinase (Jak) family of molecules that then acts on the signal transducers and activators of transcription (STAT) family of proteins. Specific Jak proteins associate with the cytoplasmic domains of cytokine receptor. When the receptor is activated by binding the cytokine, the Jak phosphorylates its respective STAT protein, causing the STAT to dimerize, and translocate into the nucleus where it then initiates new gene transcription. The essential role of Jak and STAT proteins in immune regulation is seen in individuals with inherited deficiency of these molecules (see chapter 12). Jak3 interacts with the γc protein, a subunit of several cytokine receptors including the receptors for IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21. Deficiency of the autosomally encoded Jak3 protein causes autosomal recessive severe combined immune deficiency (SCID).

All of the TLR proteins are transmembrane molecules, some expressed on the plasma membrane of the cell where they can interact with extracellular triggering molecules, and some expressed on intracellular membranes where they can interact with structures on intracellular microbes and viruses. Another set of pattern recognition molecules, designated NLR, has also been identified. These molecules are cytosolic and appear to interact with soluble intracellular ligands. Like the TLR, the NLR are characterized by the presence of leucine-rich repeat structures that are thought to contribute to their ability to bind to conserved microbial structures. The NLR can also recognize endogenous signals of cellular damage, such as uric acid crystals. Over 20 NLR-encoding genes have been identified in the human genome. Most are characterized by the presence of a C-terminal leucine-rich repeat domain that is thought to interact with microbial structures, a central nucleotide-binding oligomerization domain that is used to form multimeric complexes of the NLR, and an N-terminal effector domains that allow the NLR to recruit a class of intracellular cysteine proteinases (caspases) that activate the cellular apoptosis pathways or that activate the NF-kB transcription factor to induce a broad pro-inflammatory response.

The second activation pathway, the Alternative Pathway of Complement Activation, is activated without antibody by microbial structures that neutralize inhibitors of spontaneous complement activation. This activation pathway can deposit >105 molecules of C3b on a single bacterium in less than 5 minutes. C3b deposited in this way then triggers the MAC and also enhances phagocytosis and killing.

The third activation pathway is triggered by microbial cell wall components containing mannans and is called the Lectin Pathway of Complement Activation.

The effector mechanism of complement is potent and recruits intense local inflammation. There are several plasma proteins (factor H, C4 binding protein) and membrane proteins (complement receptors 1–4, decay accelerating factor, membrane co-factor protein) that inhibit the complement activation pathways to prevent unwanted damage to host tissues.

Rolling cells can then be induced to arrest and adhere firmly to the endothelium by interactions between integrins on the leukocyte surface and Ig domain cell adhesion molecules on the endothelial cells. Integrins are heterodimers of one α and one β chain. Key integrins for leukocyte adhesion are LFA1 (CD11a/CD18, αLβ2), VLA4 (CD49d/CD29, α4β1), and Mac-1 (CD11b/CD18, αMβ2) that bind to the Ig domain cell adhesion molecules ICAM-1, VCAM-1, and ICAM-1/C3b respectively. Binding of leukocytes to endothelial cells is enhanced by the expression of chemokines by the endothelial cells or by underlying damaged cells and tissues (see chapter 5).

After an immune response is completed, the majority of antigen responsive cells must be removed in order to prepare for the next immune challenge faced by the organism. Removal of effector cells without causing inflammation and tissue damage is best achieved by inducing the unwanted cells to undergo apoptosis. Molecules of the TNF family provide strong signals for the apoptotic programmed cell death pathway. TNF, signaling through the type I TNF receptor, induces death in tumor cells and at sites of ongoing inflammation. An alternative apoptosis-inducing receptor, Fas, is more specifically involved in regulatory apoptotic events. Fas, for example, transmits important apoptotic signals during thymic T cell selection.,

The goal of a properly regulated immune response is to protect the host from pathogens and other environmental challenges without causing damage to self-tissues. In the case of infection with viruses or intracellular bacteria and parasites, it is often impossible to eradicate the pathogen without destroying the infected cells. In cases like this, the use of cellular apoptosis as a mechanism for removing infected cells provides an elegant way to reduce damage to nearby uninfected cells. Infected cells that undergo apoptosis are generally fragmented into membrane-enclosed vesicles that can be taken up by healthy phagocytic cells and digested so as to eliminate both the potentially inflammatory contents of the infected cell and also the microbe that was multiplying inside the cell.

Some degree of local inflammation is, however, often an important part of an effective host immune response. The key elements of inflammation are part and parcel of the host’s mobilization of its defense and repair responses. When inflammation is modest and controlled, normal tissue architecture and function can be restored after the pathogen or toxin has been eliminated. If the inflammatory response is excessively severe, however, there is danger of lasting tissue damage, and the development of fibrosis during the resolution of the inflammatory state.

Perhaps more puzzling are conditions in which tissue inflammation appears to develop without any underlying infectious or noxious stimulus. Prominent in these are autoimmune diseases and atopic illnesses. These disorders appear to represent a fundamental misdirection of the immune response, resulting in tissue damage when no real danger was present. The growing spectrum of autoimmune diseases appears to represent a breakdown in self-tolerance. This leads to the induction of both cellular and humoral immune responses against components of self-tissues. Usually, both the cellular and humoral aspects of these pathological responses have features of a Th1-type or Th17-type CD4 T cell response, suggesting that defective regulation of either T cell differentiation or activation underlies the response.reg cells in controlling all aspects of the CD4 T cell response, and the observation that congenital absence of Treg cells (as in the immune dysregulation, polyendocrinopathy, enteropathy, X-linked [IPEX] syndrome) leads to development of an aggressive autoimmune statereg function may underlie all autoimmune and atopic diseases. While disordered Th1, Th17 and Th2 responsiveness is a major manifestation of these illnesses, the disorders do not simply represent a predisposition to over-polarization of the CD4+ T cell response. Epidemiological studies have shown that the presence of atopy shows little protection against development of the Th1/Th17-predominant illness rheumatoid arthritis.

A special situation in which tolerance is modulated in a physiological way concerns the suppression of the maternal immune response to permit the maintenance of the semi-allogeneic fetus and placenta in the setting of normal pregnancy. Recent studies have demonstrated that in mid-gestation human fetuses, 20–25% of the CD4 T cells in lymph nodes and spleen have a Treg phenotype and levels of TGFβ were remarkably high in these lymphoid organs.reg cells returned to normal shortly after delivery. Interestingly, spontaneous abortion has been associated with loss of normal pregnancy-associated immune suppression.reg function do not constitute the entire mechanism underlying the tolerance of pregnancy. Other studies have shown very high levels of expression of galectin-1, an immunoregulatory glycan-binding protein, in fetal tissues, and loss of galectin-1 in failing pregnancies.

22. Momburg F, Tan P. Tapasin-the keystone of the loading complex optimizing peptide binding by MHC class I molecules in the endoplasmic reticulum. Mol Immunol.2002;39:217–233. [PubMed]

75. Tangye SG, Cook MC, Fulcher DA. Insights into the role of STAT3 in human lymphocyte differentiation as revealed by the hyper-IgE syndrome. J Immunol.2009;182:21–28. [PubMed]

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The integrity of the DNA structure for cell viability is underscored by the vast amounts of cellular machinery dedicated to ensure its accurate replication, repair, and storage. Even still, mutations within the DNA are a fairly common event.

When a mutation is caused by an environmental factor or a chemical agent, that agent is called a mutagen. Typical mutagens include chemicals, like those inhaled while smoking, and radiation, such as X-rays, ultraviolet light, and nuclear radiation. Different mutagens have a different modes of damaging DNA and are discussed further in the next section. It is important to note that DNA damage, in and of itself, does not necessarily lead to the formation of a mutation in the DNA. There are elaborate DNA repair processes designed to recognize and repair different types of DNA lesions. Fewer than 1 in 1,000 DNA lesions will actually result in a DNA mutation. The processes of DNA damage recognition and repair are the focus of later sections within this chapter.

Chromosomal alterations are mutations that change chromosome structure or number. They occur when a section of a chromosome breaks off and rejoins incorrectly or does not rejoin at all. Possible ways these mutations can occur are illustrated in the figure below. Chromosomal alterations are very serious. They often result in the death of the cell or organism in which they occur. If the organism survives, it may be affected in multiple ways. An example of a human chromosomal alteration is the mutation that causes Down Syndrome. It is a duplication mutation that leads to developmental delays and other abnormalities. It occurs when the individual inherits an extra copy of chromosome 21. It is also called trisomy (“three-chromosome”) 21. Thus, large-scale mutations in chromosomal structure include: (1) Amplifications (including gene duplications) where repetition of a chromosomal segment or presence of extra piece of a chromosome broken piece of a chromosome may become attached to a homologous or non-homologous chromosome so that some of the genes are present in more than two doses leading to multiple copies of all chromosomal regions, increasing the dosage of the genes located within them, (2) Deletionsof large chromosomal regions, leading to loss of the genes within those regions, and (3) Chromosomal Rearrangements such as translocations (which interchange of genetic parts from nonhomologous chromosomes), insertions (which insert segments of one chromosome into another nonhomologous chromosome), and inversions(which invert or flip a section of a chromosome into the opposite orientation)(Figure 12.1).

Point mutations may have a wide range of effects on protein function (Table 12.1 and Figure 12.2). As a consequence of the degeneracy of the genetic code, a point mutation will commonly result in the same amino acid being incorporated into the resulting polypeptide despite the sequence change. This change would have no effect on the protein’s structure, and is thus called a silent mutation. A missense mutation results in a different amino acid being incorporated into the resulting polypeptide. The effect of a missense mutation depends on how chemically different the new amino acid is from the wild-type amino acid. The location of the changed amino acid within the protein also is important. For example, if the changed amino acid is part of the enzyme’s active site or greatly affects the shape of the enzyme, then the effect of the missense mutation may be significant. Many missense mutations result in proteins that are still functional, at least to some degree. Sometimes the effects of missense mutations may be only apparent under certain environmental conditions; such missense mutations are called conditional mutations. Rarely, a missense mutation may be beneficial. Under the right environmental conditions, this type of mutation may give the organism that harbors it a selective advantage. Yet another type of point mutation, called a nonsense mutation, converts a codon encoding an amino acid (a sense codon) into a stop codon (a nonsense codon). Nonsense mutations result in the synthesis of proteins that are shorter than the wild type and typically not functional (Summarized in Figure 12.3).

Smaller scaledeletions and insertions also cause various effects. Because codons are triplets of nucleotides, insertions or deletions in groups of three nucleotides may lead to the insertion or deletion of one or more amino acids and may not cause significant effects on the resulting protein’s functionality. However, frameshift mutations, caused by insertions or deletions of a number of nucleotides that are not a multiple of three are extremely problematic because a shift in the reading frame results (Figure 12.3). Because ribosomes read the mRNA in triplet codons, frameshift mutations can change every amino acid after the point of the mutation. The new reading frame may also include a stop codon before the end of the coding sequence. Consequently, proteins made from genes containing frameshift mutations are nearly always nonfunctional.

The majority of mutations have neither negative nor positive effects on the organism in which they occur. These mutations are called neutral mutations. Examples include silent point mutations, which are neutral because they do not change the amino acids in the proteins they encode.

Mutations have occurred in bacteria that allow the bacteria to survive in the presence of antibiotic drugs. The mutations have led to the evolution of antibiotic-resistant strains of bacteria.

Harmful mutations can also occur. Imagine making a random change in a complicated machine such as a car engine. The chance that the random change would improve the functioning of the car is very small. The change is far more likely to result in a car that does not run well or perhaps does not run at all. By the same token, any random change in a gene’s DNA is more likely to result in the production of a protein that does not function normally or may not function at all, than in a mutation that improves the function. Such mutations are likely to be harmful. Harmful mutations may cause genetic disorders or cancer.

Illnesses caused by mutations that occur within an individual, but are not passed on to their offspring, are mutations that occur in somatic cells. Cancer is a disease caused by an accumulation of mutations within somatic cells. It results in cells that grow out of control and form abnormal masses of cells called tumors. It is generally caused by mutations in genes that regulate the cell cycle, DNA repair, angiogenesis, and other genes that favor cell growth and survival. Because of the mutations, cells with the mutated DNA have evolved to divide without restrictions, hide from the immune system, and develop drug resistance.

DNA damage, due to environmental factors and normal metabolic processes inside the cell, occurs at a rate of 1,000 to 1,000,000 molecular lesions per cell per day. While this constitutes only 0.000165% of the human genome’s approximately 6 billion bases (3 billion base pairs), if left unrepaired can cause mutations in critical genes (such as tumor suppressor genes) can impede a cell’s ability to carry out its function and appreciably increase the likelihood of tumor formation and disease states such as cancer.

The vast majority of DNA damage affects the primary structure of the double helix; that is, the bases themselves are chemically modified. These modifications can, in turn, disrupt the molecules’ regular helical structure by introducing non-native chemical bonds or bulky adducts that do not fit in the standard double helix. Unlike proteins and RNA, DNA usually lacks tertiary structure and therefore damage or disturbance does not occur at that level. DNA is, however, supercoiled and wound around “packaging” proteins called histones (in eukaryotes), and both superstructures are vulnerable to the effects of DNA damage.

As mentioned previously, increased levels of 8-oxo-dG in a tissue can serve as a biomarker of oxidative stress. Furthermore, increased levels of 8-oxo-dG are frequently found associated with carcinogenesis and other disease states (Figure 12.5). During the replication of DNA that contains 8-oxo-dG, adenine is most often incorporated across from the lesion. Following replication, the 8-oxo-dG is excised during the repair process and a thymine is incorporated in its place. Thus, 8-oxo-dG mutations typically result in a G to T transversion.

AP sites can be formed by spontaneous depurination, but also occur as intermediates in base excision repair, the repair process described in section 12.5.If left unrepaired, AP sites can lead to mutation during semiconservative replication. They can cause replication fork stalling and are often bypassed by translesion synthesis, that is discussed in greater detail in section 12.8. In E. coli, adenine is preferentially inserted across from AP sites, known as the “A rule”. The situation is more complex in higher eukaryotes, with different nucleotides showing a preference depending on the organism and environmental conditions.

Benzo[a]pyrene is a polycyclic aromatic hydrocarbon that forms during the incomplete combustion of organic matter at temperatures between 300°C (572°F) and 600°C (1,112°F). The ubiquitous compound can be found in coal tar, tobacco smoke and many foods, especially grilled meats. Benzo[a]pyrene is actually a procarcinogen that needs to be biologically activated by metabolism before it forms a reactive metabolite (Figure 12.8) Normally, when the body is exposed to foreign molecules, it will start a metabolic process that makes the molecule more hydrophilic and easier to remove as a waste product. Unfortunately, in the case of benzo[a]pyrene, the resulting metabolite is a highly reactive epoxide that forms a bulky adduct preferentially with guanine residues in DNA. If left unrepaired, during DNA replication an adenine will usually be placed across from the lesion in the daughter molecule. Subsequent repair of the adduct will result in the replacement of the damaged guanine base with a thymine, causing a G –> T transversion mutation.

UV light can cause molecular crosslinks to form between two pyrimidine residues, commonly two thymine residues, that are positioned consecutively within a strand of DNA (Figure 12.10). Two common UV products are cyclobutane pyrimidine dimers (CPDs) and 6–4 photoproducts. These premutagenic lesions alter the structure and possibly the base-pairing. Up to 50–100 such reactions per second might occur in a skin cell during exposure to sunlight, but are usually corrected within seconds by photolyase reactivation or nucleotide excision repair. Uncorrected lesions can inhibit polymerases, cause misreading during transcription or replication, or lead to arrest of replication. Pyrimidine dimers are the primary cause of melanomas in humans.

Ionizing radiation such as that created by radioactive decay or in cosmic rays causes breaks in DNA strands (Figure 12.10a). Low-level ionizing radiation may induce irreparable DNA damage (leading to replicational and transcriptional errors needed for neoplasia or may trigger viral interactions) leading to premature aging and cancer. Chemical agents that form crosslinks within the DNA, especially interstrand crosslinks, can also lead to DNA strand breaks if the damaged DNA undergoes DNA replication. Crosslinked DNA can cause topoisomerase enzymes to stall in the transition state when the DNA backbone is in the cleaved state. Instead of relieving supercoiling and resealing the backbone, the stalled topoisomerase remains covalently linked to the DNA in a process called abortive catalysis. This leads to the formation of a single stranded break in the case of Top1 enzymes or double stranded breaks in the case of Top2 enzymes. DNA double strand breaks due to topoisomerase stalling can also occur during the transcription of DNA (Figure 12.11). In fact, abortive catalysisand the formation of DNA strand breaks during transcriptional events may serve as a damage sensor within the cell and help to instigate DNA damage response signaling pathways that initiate DNA repair processes.

Genetic damage produced by either exogenous or endogenous mechanisms represents an ongoing threat to the cell. To preserve genome integrity, eukaryotic cells have evolved repair mechanisms specific for different types of DNA Damage. However, regardless of the type of damage a sophisticated surveillance mechanism, that elicits DNA damage checkpoints, detects and signals its presence to the DNA repair machinery. DNA damage checkpoints have been functionally conserved throughout eukaryotic evolution, with most of the relevant players in the checkpoint response highly conserved from yeast to humans. Checkpoints are induced to delay cell cycle progression and to allow cells time to repair damaged DNA prior to DNA replication (Figure 12.12). Once the damaged DNA is repaired, the checkpoint machinery triggers signals that will resume cell cycle progression. Within cells, multiple pathways contribute to DNA repair, but independent of the specific repair pathway involved, three phases of checkpoint activation are traditionally identified: (1) Sensing of damage, (2) Activating the signaling cascade, and (3) Switching on downstream effectors. The sensor phase recognizes the damage and activates the signal transduction phase to block cell cycle progression and select the appropriate repair pathway.

In multicellular organisms, the response to DNA damage can result in two major physiological consequences: (1) Cells can undergo cell cycle arrest, repair the damage and re-enter the cell cycle, or  (2) cells can be targeted for cell death (apoptosis) and removed from the population.  The cell cycle process is highly conserved and precisely controlled to govern the genome duplication and separation into the daughter cells. The cell cycle consists of four distinct and ordered phases, termed G0/G1 (gap 1), S (DNA synthesis), G2 (gap 2), and M (mitosis). Multiple checkpoints exist within each stage of the cell cycle to ensure the faithful replication of DNA in the S phase and the precise separation of the chromosomes into daughter cells. The G1 and G2 phases are critical regulatory checkpoints, whereby the restriction point between the G1 and S phase determines whether the cells enter the S phase or exit the cell cycle to halt at the G0 phase. The cell cycle progression requires the activity cyclin-dependent kinases (CDKs), a group of serine/threonine kinases. CDKs are activated when they form complexes with cyclin regulatory proteins that are expressed specifically at different stages of the cell cycle. Cyclins bind to and stabilize CDKs in their active conformation. The formation of cyclin/CDKs controls the cell-cycle progression via phosphorylation of the target genes, such as tumor suppressor protein retinoblastoma (Rb).

During DNA damage, the cell cycle is arrested or blocked by the action of cyclin-dependent kinase inhibitors. As noted in Figure 12.12, this is a complicated signal transduction cascade that has a number of downstream effects. A primary function of cell cycle arrest is that CDK inhibition allows time for DNA repair before cell-cycle progression into S-phase or mitosis. As shown in Figure 12.12, two major cell-cycle checkpoints respond to DNA damage; they occur pre- and post-DNA synthesis in the G1 and G2 phases, respectively, and impinge on the activity of specific CDK complexes. The checkpoint kinases phosphatidylinositol 3-kinase (PI3K)-like protein kinases (PI3KKs) ataxia telangiectasia and Rad3-related (ATR) or ataxia telangiectasia mutated (ATM) protein, and the transducer checkpoint kinases CHK1 (encoded by the CHEK1 gene) and CHK2 (encoded by the CHEK2 gene) are key regulators of DNA damage signaling. The DNA damage signaling is detected by ATM/ATR, which then phosphorylate and activate CHK2/CHK1, respectively. The activated CHK2 is involved in the activation of p53, leading to p53-dependent early phase G1 arrest to allow time for DNA repair. The activation of p53 induces the expression of the Cyclin-Dependent Kinase Inhibitor (CKI) p21CIP1 gene, leading to inhibition of cyclin E/CDK2 complexes and subsequent upregulation of DNA repair machinery.

If the DNA repair cannot be completed successfully or the cells cannot program to respond to the stresses of viable cell-cycle arrest, the cells face the fate of apoptosis induced by p53. The activated CHK1 mediates temporary S phase arrest through phosphorylation to inactivate CDC25A, causing ubiquitination and proteolysis. Moreover, the activated CHK1 phosphorylates and inactivates CDC25C, leading to cell-cycle arrest in the G2 phase. The active CHK1 also directly stimulates the phosphorylation of WEE1, resulting in enhancing the inhibitory Tyr15 phosphorylation of CDK2 and CDK1 and subsequent cell-cycle blocking in G2 phase. The activity of WEE1 can also be stimulated by the low levels of CDK activity in G2 cell-cycle phase. The SAC, also known as the mitotic checkpoint, functions as the monitor of the correct attachment of the chromosomes to the mitotic spindle in metaphase, which is regulated by the TTK protein kinase (TTK, also known as monopolar spindle 1 (MPS1)). The activation of SAC transiently induces cell-cycle arrest via inhibiting the activation of APC/C. In order to establish and maintain the mitotic checkpoint, the TTK recruits many checkpoint proteins to kinetochores during mitosis via phosphorylating its substrates to ensure adequate chromosome segregation and genomic integrity. In this way, the genomic instability from chromosome segregation defects is protected by SAC. Once the SAC is passed, the APC/C E3 ligase complex stimulates and tags cyclin B and securin for ubiquitin-mediated degradation, leading to the initiation of mitosis. In a word, the checkpoints offer a failsafe mechanism to ensure the genomic integrity from the parental cell to daughter cell. The signal transduction cascade of checkpoint activation eventually converges to CDK inhibition, which indicates the CDK function as a key driver of cell-cycle progression.

Figure 12.12 Cell Fates Following DNA Damage. Cell cycle checkpoints are induced by DNA damage, shown in red. Cyclin-Dependent Kinase Inhibitors (CIP/KIP), shown in purple, block cell cycle progession in all phases of the cell cycle (G1, S, G2, or M) following DNA damage by inhibiting cyclin-dependent kinase complexes (shown in green). Signaling cascades activated in response to DNA damage also elicit DNA repair pathways, or if the DNA damage is severe, programmed cell death (Apoptosis) will be activated.

In addition to blocking cell cycle progression, DNA damage sensors also activate DNA repair mechanisms that are specific for the type of damage present. For example, single stranded DNA breaks are repaired primarily by Base Excision Repair, bulky DNA adducts and crosslinks are repaired by Nucleotide Excision Repair, and smaller nucleotide mutations, such as alkylation are repaired by Mismatch Repair. Cells also have two major mechanisms for repairing Double-Strand-Breaks (DSBs). They include Non-Homologous End-Joining (NHEJ) and Homologous Recombination (HR). If damage is too extensive to be repaired, apoptotic pathways will be elicited. In the following sections, details about the major DNA repair pathways will be given.

DNA mismatch repair (MMR) is a highly conserved DNA repair system (Table 12.2) that greatly contributes to maintain genome stability through the correction of mismatched base pairs and small modifications of DNA bases, such as alkylation. The major source of mismatched base pairs is replication error, although it can arise also from other biological processes. Thus, the MMR machinery must have a mechanism for determining which strand of the DNA is the template strand and which strand has been newly synthesized. In E. coli, methylation of the DNA is a common post-replicative modification that occurs.  Thus, in newly synthesized DNA, the unmethyl