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Immune Evasion by bacteria

How do bacteria circumvent the immune system? Obviously, if the immune system successfully recognises, consumes and destroys all of the organisms that are present in any given infection, then that organism has failed to establish itself in the body, and thus cannot cause disease. However, the immune system does not always succeed in its task. Often the reason for this failure is that the invading organism has evolved a strategy for evading or suppressing the hosts immune response to that organism. Many different strategies have evolved in different organisms; below is presented a list of some of those strategies. Strategies directed against acquired immunity

Molecular mimicry. Organisms that employ this strategy genetically resemble parts of the hosts own body. The body is less likely to allow the existence of immune antibodies that act against the body itself (i.e. autoimmunity), since that can be highly dangerous to the body. By mimicking the genetic/chemical makeup of the body, organisms can exist without an immune response being activated against them. The bacterium Bacteroides, for example, can avoid evoking an immune response in mice. There are several bacteria that mimic parts of the human body, but these usually give rise to an immune response. Since the resultant anibodies act against both the foreign organism and the bodys own tissues, this has the effect of making the body mount an immune response against itself. Mycobacterium tuberculosis, Mycobacterium leprae, Treponema pallidum, Mycoplasma pneumoniae are all organisms that causeautoimmunity in humans. Mycobacterium paratuberculosis has been demonstrated to use this strategy in animals. Suppression of antibodies. In this strategy, the invading organism employs the strategy: the best form of defense as attack. The organisms in this category target those cells of the immune system that specifically react against them. By disabling those cells, the organism prevents the body from mounting an immune response against the invader. Two notable bacteria that suppress the bodies reaction against them are Mycobacterium leprae, the cause of the disease leprosy, and Mycobacterium tuberculosis, the cause of the disease tuberculosis. In both cases, the Interleukin-2 response to the bacteria is reduced. In lepromatous leprosy, it has been shown that suppressor T cells, taken from the skin lesions caused by the disease, inhibit the responses of other T cells to Mycobacterium leprae antigens. Hiding inside cells. Many bacteria avoid an immune response by hiding inside the cells of the immune system. By doing so, they do not present antigens that will evoke an immune response. They multiply inside these cells, and then further invade the body when they are greater in number. Among the bacteria that use this strategy are Brucella, Listeria, Mycobacterium leprae andMycobacterium tuberculosis, all of whom infect macrophages, the cells that are normally responsible for destroying invading bacteria. Mycobacterium leprae can also invade and inhabit cells that are not a part of the immune system, e.g. skin cells. By releasing antigen into the bloodstream. Normally, the antigen of an invading organism that is recognised by the body exists on the cell wall of that organism. Some organisms, however, release these antigens from their cell walls to float freely in the bloodstream, where eventually they will meet their antibody, and will bind to them, thus rendering those antibodies ineffective. To provide an analogy, if the immune system were to recognise only the "face" of an organism, then organisms that use this strategy not only shed their "faces", but also produce as many "faces" as they can, which act

as decoys and confuse the immune system. This leaves the rest of the organism freedom to move about unhindered. Strategies directed against Phagocytes As you remember, the Phagocytes, i.e. the Macrophages and Microphages, are the cells responsible for consuming and destroying invading bacteria. Many microbial strategies for survival involve action against the Phagocytes. Strategies include

Inhibiting Chemotaxis. Chemotaxis is the chemical process by which thePhagocytes are led to the site of infection, so that they can begin their task. Some bacteria, such as Staphylococcus Aureus, produce toxins which inhibit the movement of Phagocytes, which hinders them in their journey. Inhibiting Phagocytosis. In order to Phagocytose (i.e. swallow and consume) bacteria, phagocytes must first grip onto the invader, and engulf them. Some bacteria evade Phagocytosis by not presenting anything for the phagocytes to grip onto. Killing the Phagocyte. Some bacteria are capable of releasing toxins that are lethal to Phagocytes. So instead of the invading bacteria being destroyed, the defending phagocytes are themselves destroyed. Among the bacteria that are capable of this strategy are Mycobacterium tuberculosis, Streptococcus pyogenes, some Staphylococci, and Bacillus Anthracis (the bacterium that causes the disease Anthrax). Colonising the Phagocyte. This highly successful strategy involves the invading bacteria allowing themselves to be Phagocytosed, but resisting being killed once they are inside the Phagocyte. Many types of bacteria use Macrophages as sites of sanctuary, where they can multiply without interference from other cells of the immune system. As mentioned above, bacteria that use this strategy include Mycobacterium leprae and Mycobacterium tuberculosis.

The Pathology of Mycobacterial Diseases.

Well known human diseases that are caused by mycobacteria are tuberculosis, caused by Mycobacterium tuberculosis, and leprosy, caused by Mycobacterium leprae. In common with all mycobacterial diseases (known as mycobacterioses), these diseases come in two forms, the contained form and the aggressive form, known as the "polar" forms of the disease. The Contained form. The contained form of leprosy is known as tuberculoid leprosy and the contained form of tuberculosis is simply known as the contained form of tuberculosis. People who suffer from the contained forms of mycobacterioses are known to mount only a "Cell Mediated Immunity" (CMI) response to the infecting mycobacterium. This CMI response is activated only by T cells. Sufferers do not mount a "Humoral" response to the invading mycobacterium. This most recognisable symptom of the contained form is the formation of Granulomas. Granulomas are formed when an invading mycobacterium, possibly living inside an infected host cell, is surrounded by T cells. First a layer of CD4 T cells surrounds the mycobacterium, and then a layer of CD8 T cells surrounds the layer of CD4 T cells. This has the effect of forming a hard lump (known as a "tubercle" in tuberculosis) around the mycobacterium, from which it cannot escape to further infect the body. Tissue damage around these granulomas can be extensive, especially if they occur in highly sensitive areas of the body, such as nerve cells, or the lymph nodes. If formed in the intestines, they lead to the formation of "strictures", which restrict free

passage of food through the intestines. In "tuberculoid leprosy" sufferers, these granulomas are seen as lumps in the skin, and can restrict the flow of blood to extremities (fingers, toes, etc.) The contained forms of mycobacterioses are characterised by the presence of very few numbers of the infecting mycobacteria compared to the damage caused. The Aggressive form. The aggressive form of leprosy is known as "lepromatous leprosy", and the aggressive form of tuberculosis is known as "miliary tuberculosis". People who suffer from the aggressive forms of mycobacterioses are known to mount only a "Humoral" response to the infecting mycobacterium. This Humoral response is activated only by B cells. They do not mount a "Cell Mediated Immunity" (CMI) response to the invading mycobacterium. Common symptoms of the aggressive forms of mycobacterioses are perforation, fistulisation of the infected organs and abscess formation. Perforation leads to holes in the infected organs, leading to a breach of the integrity of body cavities. A fistula is a growth of tissue from one organ to another. For example, a fistula may connect the intestines to the bladder, or to the external skin of the sufferer. If untreated, miliary tuberculosis will almost certainly result in the death of the sufferer. The aggressive forms of mycobacteria are characterised by the presence of large numbers of the infecting mycobacteria, which rage uncontrolled in the infected host. Autoimmunity. A possible cause of these failures of the CMI response or of the humoral response is autoimmunity. Some proteins (known as antigens) which make up mycobacteria are identical to proteins that exist in the human body. The immune system uses these antigens to combat infections. Both T cells and B cells recognize antigens, and form other proteins (antibodies) that bind these antigens, thus rendering them harmless, and they can then be removed from the body (the antibody-antigen pair is known as an immune complex). If antibodies are formed to antigens that are identical to human proteins (known as self-reactive antigens), then those antibodies will also attack the tissues of the body that contain these antigens. These tissues may be far from the site of infection. This is how secondary inflammatory symptoms, such as arthritis or uveitis, come about. This phenomenon is known autoimmunity. In some individuals, the body will not allow such self-reactive antibodies to exist, because they are harmful to the body, and destroys them. This is known as an anergy reaction. A full spectrum of reactions. In practice, mycobacteriosis patients seldom exhibit the extreme contained or aggressive forms described above, but exhibit a form that is a hybrid of the two. For example,"borderline leprosy" is a form of leprosy that exhibits symptoms from both tuberculoid and lepromatous leprosy. Immunology. Immunology research, where researchers seek to measure the bodies immune response to various bacterial antigens, are highly specific. Most research looks for an humoral response or for a CMI response. As can be seen from the above, a search for a CMI (T cell) response to mycobacterial antigen will show a response in sufferers of the contained form, but will show no response in sufferers of the aggressive form. Likewise, a search for an humoral (B cell) response will show a response in sufferers of the aggressive form, but no

response in sufferers of the contained form. Statistical analysis of results of such searches, without first dividing the patients into two groups that represent the different forms, will yield random results.

Innate immune response Initial responses in Macrophages and dendritic cells: TLR-2 mediates NFkB, and TNF secretion, and binds to the M. leprae and M. tuberculosis wall (2). Some researchers think that the effective functioning of TLR-2 is necessary to the development of the less severe tuberculoid leprosy, in contrast to a systemic lepromatous disease manifestation (2). In lepromatous leprosy, these studies have shown that a significant number of patients have a defective TLR-2 receptor, which then prohibits any cellular immune response from starting (2). Such a lack of cellular immunity apparently causes the M. leprae bacterium to proliferate much more efficiently in the human body (2). TLR-4 may also play a role in M. leprae binding, but to what extent, researchers are not sure (4). The presence of CD14 on the APC surface increases cell mediated responses to M. leprae up to two-fold, but is not necessary for the initiation of the signaling cascade. (2). Studies also show that in lepromatous leprosy, cytokines and chemokines are not released by macrophages - and neither are MHC-I or II significantly expressed (3, 5)! In fact, this inhibition of chemical release may serve instead to enhance bacterial growth (5).

When macrophages (or dendritic cells) do get activated by infection with M. leprae, a number of receptors and inflammatory molecules are upregulated. The interleukins IL-1, IL-2 IL-12, and IL-18 (which acts on both NK and T-cells) are expressed (5). TNF-alpha is secreted in order to activate NK cells (5).

M. leprae induces apoptosis in macrophages in a dose-dependent manner (6). Apparently, this is correlated with high levels of TNF-alpha, as well as various apoptotic receptors (4). It is thought that this apoptosis helps to inhibit bacterial proliferation of the mycobacterium since mycobacteria are not efficient at replicating outside of the cell (4). In fact, researchers claim that this may be one of the main differences between tuberculoid and lepromatous leprosy - this initial clearing of bacterial load through macrophage apoptosis (4).

The cell membrane of M. leprae is highly antigenic, and dendritic cells recognize it readily (6). Upon recognition, the dendritic cells produce IL-12 (6). MMP II is a protein on the surface of M. leprae that stimulates expression of HLA-ABC, HLA-DR,

CD86, and CD83 antigens in dendritic cells, and also induces maturation (6). All of this indicates that MMP-II is the protein on M. leprae that binds to TLR-2 on the APC surface (6). Studies also show that the expression of CD1 by dendritic cells is also critical to the activation of T-cells in the later adaptive immune response of the body (7). The deal is, CD8+ cells are critical to defeating M. leprae once infection has happened (3). The CD8+ cells go around and kill any M. leprae bacteria they can find using perforin and granulysin (3). This is great because they are quite efficient at getting rid of the bacteria that are killing the macrophages (3). However, the T-cells must first be activated and that is often done by dendritic cells (3). So far, dendritic cells presenting CD1+, CD83+, and the CD40 ligand have been found in areas of M. leprae infection, indicating that they do, in fact, play a significant role in activating T-cell immunity (3). So far, it is difficult to determine how effective dendritic cells are at activating T-cells specifically during M. lepraeinfection (3). Studies have shown that infection with M. leprae downregulates the presentation of both MHC-I and II on the surface of dendritic cells, in addition to a lack of significant upregulation of either CD83+ or IFN-gamma (3). Initial Responses in neutrophils and NK cells: Both neutrophils and NK cells are very important in inducing further immune response after infection with M. leprae (1). NK cells are required to induce IFN-gamma production, which further activates macrophages and dendritic cells (1). NK cells then help to activate T-cells and promote the differentiation of Th1 cells - a vital part of the defense system againstM. leprae infection (1). The activity of NK cells at the site of infection is generally used as a determinant of how successful host defense is, as they serve to both kill infected cells and also heavily activate other members of both the innate and adaptive immune system (1). NK cells also secrete IL-13, though in smaller amounts compared to T-cells (1). This is interesting, because IL-13 is a known cytokine inhibitor, and generally stops any further IFN-gamma production - and therefore essentially stops the inflammatory process (1).

Humoral immunity Humoral immunity involves the interaction of circulating antibodies in blood serum in addition to the actions of B-cells as activated by Th2-cytokine profiled T-cells. In other words, humoral immunity involves the adaptive portion of the immune system response that which takes a period of days to take effect after infection has begun.

The immunoglobulin complex (1 - Janeway et al, 2005 Figure1-17)

Activation of B-cells: B-cells are only significantly activated during the immune response by the cytokines produced by Th2 T-cells: specifically, IL-4, Il-5, and Il-10 (2). Furthermore, a Th2-predominant T-cell response implies an ineffectual immune response to the pathogen and results in a severe, often fatal form of leprosy: lepromatous leprosy. This is likely because of the mycobacteriums unique lifecycle that requires it to replicate inside host cells instead of

free in tissue or blood. Having to replicate inside of cells prevents much recognition of the bacterial cell wall and limits any recognition of infection to somatic cell presentation of bacterial epitopes on MHC-I. Interaction of B-cells and M. leprae: There is not much information on M. lepraes interaction with B-cells. It does seem clear, however, that in the specific case of M. leprae, B-cell activation does not play a significant role in clearing the bacteria from the body. As evidenced, IgG and IgM blood serum levels are high during lepromatous leprosy infection, while the immunoglobulin levels are anywhere from high to undetectable levels during tuberculoid leprosy infection, depending on the activity of the disease in the specific patient (3). In general, however, this study found that patients with lepromatous leprosy had a stronger IgG and IgM response than patients with tuberculoid leprosy (3). This trend is indicative of an unnecessary antibody response, given that the milder tuberculoid leprosy causes a wide range of reactions, including no immunoglobulin secreted at all (3). During infection of M. avium (a mycobacterium related to M. leprae which infects the gastrointestinal tract of cattle), an increasing humoral immune response to the pathogen is paired with higher bacterial shedding in the feces and eventually death (4). Researchers further posit that this trend found in M. avium can be applied to similar mycobacterial infections (4). Infection with M. avium proved to both lower antigen-specific B-cell levels and also to raise unspecific, circulating peripheral B-cells (4). These seemingly contradictory conclusions have not yet been fully described, and their role in promoting further infection by mycobacteria are not understood (4). Cellular response: Cell mediated immunity is the part of the immune response that involves actions of macrophages, natural killer (NK) cells, activated killer T cells (CD8+), and the actions of various cytokines. Cell mediated immunity is extremely important during the infection process of M. leprae due to the inability of B-cells to mount a useful immune response to pathogen. Th1 cellular role: Although Th1 cells are not cytotoxic, they do play a very significant role in cell-mediated immunity. In the case of M. leprae, an important facet of an effective immune response is the delineation of T-cells into Th1 and Th2 cell lines. A Th1-dominant immune response to M. leprae indicates development of tuberculoid leprosy (a milder form of leprosy) (3). Th1 T-cells secrete the cytokines Il-2 and IFN-gamma, which support delayedtype hypersensitivity to the pathogen (3). The Th1 immune response is thought to be chosen through circulation of IFN-gamma and IL-12 (3). Interestingly, IFN-gamma also inhibits the progression of T-cells into Th2 cells (3). A Th2-dominant immune response to M. leprae indicates development of lepromatous leprosy, which is discussed in the section humoral immunity.

Despite studies that show the importance of a Th1-mediated immune response, studies have shown that up to 50% of patients with various types of leprosy have a mixed cytokine profile, also known as Th0 (3). In this Tcell profile, the cells have not differentiated into Th1 or Th2 cytokine profiles (3). There are a few caveats to this fact; namely, the aforementioned studies only looked at cytokine profiles from the lymph nodes of patients with leprosy, and not closer to the site of infection or lesion (3). Previous studies have shown that T-cells nearer to the actual infection are more likely polarized into the Th1 or Th2 cytokine profile (3). Macrophage response: Since M. leprae infects macrophages, their activation proves very important for clearing the bacterial infection. Macrophages are generally activated by Th1 cells, which activate cell-mediated immunity in general (2). When the infected macrophages are inactive, M. leprae is able to evade the cellular immune response and replicates inside of the cell until it bursts. Without any signal from outside sources, macrophages are unable to mount any significant response to the bacterium, and the infection spreads largely unchecked (2). When activated through Th1 cells (and the cytokines secreted by them), macrophages then are more likely to apoptose (thereby killing the residing bacterial load) and also activate their lysosomes to fuse with any phagosomes that might be harboring bacteria (2). NK response: A Th1 predominant T-cell response usually indicates early movement of the NK cells to the site of infection (1). However, NK cells are also very active in the early stages on infection, before the adaptive immune system has time to kick in (1). In each case, NK cells are very effective at fighting M. leprae infection as long as they are both activated (most likely by Th1 cells) and can recognize the infected macrophages or Schwann cells (2).

Immune System Evasion Generally, M. leprae manages to avoid the immune system through its unique lifecycle: by replicating exclusively inside of macrophages and Schwann cells, most phagocytes and antigen-presenting cells do not encounter the pathogen. This lack of meeting causes a paucity of immune response hence, M. leprae stays mainly silent.

However, even when an immune response has begun, M. leprae can often avoid complete clearance by the immune system. By causing a predominantly Th2 cell response, M. leprae is able to avoid direct interaction with the immune system by preventing the cells that might recognize infected host cells from being activated in the first place (3). While a predominantly Th2 cell response only occurs in a fraction of leprosy cases, it is enough to provide a significant evasion system for the pathogen. These cases provide such an ineffective immune response that the bacterium is actually able to proliferate significantly more, and usually results in death (3). In tuberculoid leprosy, immune system evasion tactics are more specific, and less effective. Specifically, M. leprae has developed a mechanism to prevent host cell apoptosis by affecting a number of apoptosis-linked mRNAs (2,4). Apoptosis Prevention: M. leprae is able to upregulate the host macrophages production of Mcl-1, an anti-apoptotic gene (2). In addition, M. leprae downregulates the cells expression of the pro-apoptotic genes Bak and Bad (2). When activated, Bad forms heterodimers with Bcl-x and Bcl-2 thus switching their roles from apoptosis preventors to activators (2). A prevalence of Bad in the cell therefore makes it more prone to apoptosis. Like M. tuberculosis (which is a mycobacterium similar to M. leprae that causes tuberculosis), M. leprae is also able to prevent host macrophage apoptosis in vitro (4). While this may not be the case in vivo, it is an interesting point that the bacterium is able at least in vitro to affect the sensitivity of the host cell to apoptosis. The mycobacteria find a way to increase expression of Fas-ligand and decrease expression of the Fas receptor on the surface of the macrophages (4). This allows the macrophage to protect itself against killing by CD8+ Tcells (3). Fas ligand is usually only expressed by the CD8+ cell itself and recognizes the Fas receptor on infected cells (3). Therefore, the host macrophages increase of Fas-ligand expression serves to hide it from recognition by the CD8+ T-cell. By this mechanism, M. leprae may (in vivo) be able to evade immune system recognition by CD8+ T-cells, thereby avoiding the results of a Th1 predominant response (2).

Induction of anergy: Similarly, M. leprae infection of dendritic cells causes them to be poor inducers of T-cell activation in vitro (1). In addition, the study suggested that infected DCs that have not fully matured can induce an anergic state in the DC where it activates regulatory T-cells instead of CD4+ or CD8+ cells and therefore suppresses any previously activated specific immune response (1). Again, this study was only conducted in vitro, and it is still questionable as to whether the trend could be replicated in vivo.

15-26 Host response to viral pathogens relies heavily on T cells

With intracellular parasites, viruses and some microorganisms, cytotoxic T cells are particularly important. After activation, cytotoxic T cells attacked infected host cells killing them. The response to a viral infection is quite different from that seen in a bacterial infection. Viral infections are intracellular for the most part, while the case we described above was extracellular. This has two implications. First, much of the damage is going to occur inside infected cells. Second, elimination of the infection is going to require destruction of host cells. Therefore, the immune system needs a method of discriminating virally infected cells from healthy cells. Fortunately our bodies have just such a mechanism and it involves the MHC I molecules. In general, cell-mediated immunity is much more important for clearing a viral infection.

Immune response to influenza infection

An immune defense against a viral infection is more dependent on T cells and less dependent on antibodies. Cytotoxic T cells are important in killing virally infected cells. In step (1) influenza virus enters the cell and begins

to replicate (2). Viral proteins fill the cytoplasm (3). Most go on to form influenza virus and escape (4). The presence of viral proteins in the cytoplasm also causes the production of -interferon (5). Some viral proteins are degraded and end up being displayed in MHC I molecules (6). Passing T c cells will test the MHC I molecule presenting foreign antigen and a fraction will be activated by it. Activated T c cells are directed to differentiate into cytotoxic T cells by TH1 cells. Activated cytotoxic T cells then attack other virally infected cells displaying viral antigens in the MHC I molecules that the T cells react to. Viral antigens are also presented to B cells producing antibodies in a similar fashion to that described in Section 15-25. These antibodies attack free virus, agglutinating it and making it available for phagocytosis (9). Influenza virus is a good example to study because it illustrates many of the salient points of viral infection and is also an important human pathogen. However, remember that the specific response of the immune system is dependent upon the particular pathogen. Many of the mechanisms that we describe here come into play in other viral infections, but the exact response is always unique to the particular viral agent. Influenza is a very contagious disease that easily spreads through respiratory droplets expelled by infected individuals. For this example, imagine that an influenza sufferer has just sneezed on their hand and then opened a door, contaminating the door handle. Another person touching the handle can picked up droplets containing 100,000,000 flu viruses and a subsequent touch can transfer 1,000,000 to the nasal or oral cavity. Some of them then land at the back of the throat. Again, the low pH and unfriendly environment created by the normal flora and host proteins cause the vast majority of the viruses to be inactivated. Those that do survive bind to sialic acid-containing proteins or lipids on the surface of throat epithelial cells and enter by receptor-mediated endocytosis. A drop in the pH of the endosome causes a conformational change in the virus and the release of the eight genomic fragments of influenza virus into the cell. Up until this point, the immune system has no indication that anything is amiss. Virus begins to replicate and viral proteins accumulate in the infected cell. Some of these proteins are degraded by host cell machinery and the peptide fragments are transported into the endoplasmic reticulum, where they combine with newly synthesized MHC I molecules. The MHC I molecules loaded with foreign viral antigens now find their way to the surface of the cell. The infected cell also begins to produce and secrete -interferon. This notifies surrounding cells of viral infection and induces them to produce compounds that interfere with viral replication making further infection more difficult. Virally infected cells begin to release flu virus particles into the surrounding tissues. The presence of viral particles and the death of virally infected cells begin the process of inflammation as described for S. pyogenes infection. This causes the characteristic redness, soreness and swelling in the back of the throat and the induction of fever observed in influenza. At this point, macrophages and dendritic cells take up viruses and viral debris, process them and add to MHC II molecules for presentation to T helper cells. T H1 cells detect the presented antigen and those that match are activated and begin to secrete IL-2, which has several effects on other T and B cells responding to the infection. Mucous secretion from the intensifying inflammation begins to cause a runny nose and coughing. The release of interferon and IL1 contribute to the aches and fever associated with influenza. As the concentration of virus increases in the body, these symptoms intensify. Cytotoxic T cells (Tc cells) roaming in the tissues encounter infected cells presenting viral antigens in their MHC I molecules. Those that have TCRs that respond to the antigen are activated to begin clonal expansion and develop into active cytotoxic cells under the influence of the T H1 cells. During the next encounter with a virally infected cell, these cells again recognize the viral antigen being presented in an MHC I molecule using their TCR, but this time, they are activated to attack the cell. The T c cell binds to the infected cell and begins a destructive cycle. A number of cytokines, including -interferon and tumor necrosis factor (TNF), are secreted by the T c cell. These factors limit viral replication in the target cell and also attract phagocytes to the area. The T c cells also produce molecules that elicit a form of programmed cell death (apoptosis) in the target cell, essentially telling the cell to kill itself. Phagocytes that enter the area then clean up any remaining viral debris. As in the case of B cells, some of the Tc cells of the clonal expansion do not differentiate, but remain as memory cells in preparation for the next viral challenge by this viral strain.

Phagocytized virus is also presented to TH2 cells that respond by activating in a manner identical to that described earlier for bacterial infections. Virally infected cells also stimulate B cells that respond by clonal expansion and differentiation into plasma cells, resulting in the formation of antibodies against viral antigens. Antibodies are commonly raised against hemagglutinin and neuraminidase, two proteins on the outer surface of the virus. In most viral infections, antibody is not as important as in bacterial infections, but it does have several consequences. Antibodies bound to virus cause them to agglutinate, precipitate them out of solution and slow their spread through the body. Many effective antibodies block the receptor site of the virus and prevent its attachment to new host cells. These types of antibodies are especially useful in stopping subsequent infections by the same virus. Antibodies attached to virus also assist phagocytes in the efficient uptake of viral particles. Free virus in the body is eliminated by the action of antibodies and phagocytes and the activated T c cells destroy any virally infected cells. As the load of virus present in the body decreases, T suppressor cells help the immune response abate. After infection, subsequent attack by this strain of influenza virus is prevented by the action of T and B memory cells. rinter
THE IMMUNE SYSTEM AND PARASITIC EVASION Throughout the course of evolution, parasitic organisms have developed several methods to alter their environments so that they may survive and reproduce, thereby ensuring their propagation. These adaptive strategies can be passive or may involve active manipulation of the host's immune system through evasion, exploitation, and molecular piracy. The particular mechanisms used depend on the parasites:

life-cycle stage route of penetration microenvironment in which it is established inside the host (4)

Using Leishmania as our model parasite, we will discuss several such paradigms of parasitic immune evasion. Specifically, we will compare the intracellular evasion mechanisms of inhibition of phagosome-lysosome fusion, expression of MHC-I and II molecules, and peptide loading along with prevention of apoptosis to the extracellular mechanism of complement lysis evasion. The host's mechanisms of defense against parasites range from the relatively simple primary barrier to more elaborate mechanisms that involve a variety of cells and molecules capable of specific recognition and elimination of pathogens (6). Following penetration of a physical barrier (the body's first layer of protection), the innate immune system generates an immediate but non-specific response. This can include the mobilization of immune cells by the production of cytokines; activation of the complement cascade to identify the invader, activate immune cells, and promote the clearance of dead cells and antibody complexes; identification and removal by white blood cells; and, if unsuccessful in eliminating the pathogen, activation of the adaptive immune system via antigen presentation. In the adaptive immune response, the pathogen is identified as non-self during antigen presentation, and this generates specific responses that are tailored to maximally eliminate the pathogen itself or cells it has infected. This results in the development of immunological memory so that future attacks are met with a stronger, faster immune response (7). EXTRACELLULAR EVASION OF THE HUMORAL IMMUNE RESPONSE The extracellular portion of the Leishmania life cycle focuses on the transition from the promastigote stage (existing in the sandfly vector and outside of the host macrophage) to the amastigote stage (existing inside the host macrophage) (8). After entering the human host during the blood meal of a female sandfly, Leishmaniapromastigotes must first evade complement-mediated lysis in the bloodstream until they are engulfed by a macrophage (9). The promastigotes are considered

part of the extracellular phase of Leishmania because they have not yet developed into intracellular amastigotes. At this point, the parasite is exposed to the potentially lethal effects of the complement system. In normal humoral immune system functioning, the complement system functions to pierce the cell membrane of a pathogen, leading to its lysis. It consists of the membrane attack complex (MAC), which is made up of complement proteins. (Each protein begins with C followed by a number reflecting their order of discovery.) The MAC must first be assembled and activated before it can attack invaders, and this can be done in one of two waysthe classical pathway or the alternative pathway. In each pathway, complement proteins are activated by successive cleavages and ultimately result in the formation of the MAC. The MAC is composed of C5b-8 and acts as a receptor for membrane-disrupting C9 molecules. When C5b-8 and poly-C9 bind, the MAC forms a transmembrane pore that leads to the lysis of the target cell (10). In classical activation, complement proteins are activated by antibodies. Conversely, in the alternative pathway a variety of antigens and components of viruses or other pathogens activate complement proteins. Following complement pathway activation via the alternative pathway, MHC is also then able to insert into a pathogens cellular membrane and destroy it. Whichever pathway is used to activate the MAC, this system is essential for fighting off foreign invaders. The Leishmania parasite has devised a way to evade complement lysis so that it may go on and infect macrophages. Several hypotheses have been suggested to explain the exact mechanism. In general, each proposed mechanism prevents the proper functioning of the MAC complex, albeit at different points in the activation cascade. Most research supports the theory that lysis evasion depends on the differentiation of promastigotes into metacyclic forms in the sandfly, which are resistant to complement. However, this mechanism is specific to the Leishmania major species, where procyclic promastigotes are unable to resist complement action. Furthermore, when insect-stage procyclic promastigotes develop into infective metacyclic promastigote forms, their membrane is altered to prevent insertion. The major surface molecule of the promastigote, the lipophosphoglycan (LPG) is expressed on the parasite surface at this time and is considered a possible barrier for the insertion of the MAC C5b-C9 subunits into the parasite surface membrane because it is approximately twice as long as the form on procyclic promastigotes (11). Alternatively, development into the metacyclic form is associated with changes in membrane carbohydrates, altered motility, and enzyme activities (12). The metacyclic forms are thus highly active and are larger in size because they have more protein and less carbohydrate, which could explain why it differs from the procyclic promastigote in complement reaction. A different mechanism specific to Leishmania donovani promastigotes is the prevention of C5 convertase formation by fixing the inactive C3bi subunit on their surfaces (9). The surface glycoprotein gp63, a protease, has been reported to protect Leishmania amazonensis and Leishmania major against cellular lysis by converting C3b into C3bi, thus favoring parasite opsonization. Opsonization makes the parasite more susceptible to phagocytosis and thus less vulnerable to complement (9). Interestingly, Leishmania also exploits the complement system to increase its infectivity of host cells. It uses complement activation as a means to be phagocytized by a macrophage. Unlike other parasites such as Toxoplasma gondii, Leishmania does not use a specific receptor on the host cell to gain entry and is instead passively taken up by macrophages. It relies on complement proteins and antibody to coat its surface, which then leads to opsonization the ability of macrophages to take up opsonized (antibody-coated) particles (7). By subverting the complement system and exploiting it as a means for host cell entrance, Leishmania is able to persist within its extracellular environment.

INTRACELLULAR EVASION OF THE CELL-MEDIATED RESPONSE As mentioned earlier, Leishmania lives primarily in macrophages. Macrophages are derived from white blood cells and are found in the tissues. Their role is to clear cellular debris and pathogens from the body through phagocytosis. In normal cellular immune system functioning, the macrophage ingests a parasite into a phagosome, which fuses to one of the cell's many late endosomes and lysosomes. Parasite proteins are then degraded into short peptide fragments, which the macrophage then presents in the context of MHC class II molecules to CD4+ helper T cells, another type of white blood cell, in the lymph node. Macrophages may also display parasite peptides in the context of MHC class I molecules to CD8+ cytotoxic T cells. This activates the cytotoxic T cells to proliferate and destroy the infected macrophage through the secretion of the cytokine interferon gamma (IFN-g) (7). Alan Sher of the National Institute of Health notes that "it's kind of ironic that Leishmania decides to live in the most dangerous cell in the body (3). It is only able to do so, however, by manip ulating the macrophage's machinery in various ways. Most notably, Leishmania inhibits the macrophage proteolytic process by preventing the fusion of its own parasitophorous vacuole (a nonfusigenic version of a phagosome that lacks important host surface proteins) with the cell's lysosomes. LPG, which has been previously mentioned in the extracellular evasion of complement, is imperative in this process as well. Studies have confirmed that the LPG surface protein becomes incorporated into the phagosome membrane and somehow inhibits parasitophorous vacuole fusion with endosomes, in spite of the fact that the host cell fusion machinery remains operational during infection (14). The exact mechanism by which LPG prevents phagosome-endosome fusion is not known. LPG is also implicated in the prevention of apoptosis by Leishmania-infected macrophages. Apoptosis is a signal-dependent physiologic suicide mechanism that allows for the homeostatic balance between cell proliferation and cell death. It involves the transcription of specific genes and requires a trigger from the environment, usually exposure to or the removal of a specific growth factor or hormone. It has been shown that LPG inhibits apoptosis in bone marrow macrophages in vitro and that the parasite also induces the expression of a number of cytokine genes (15). Therefore it is thought that with the help of LPG, Leishmania-mediated inhibition of apoptosis may be related to the stimulation of cellular cytokine gene expression. Specifically, the cytokine TNF a is induced in infected cells, and acts in an autocrine manner to inhibit apoptosis with the help of additional factors such as LPG. However, if the phagolysosome does form, another parasitic surface protein, gp63, inhibits degradative phagolysosomal enzymes (16). This also ensures the survival of the parasite within the host cell. Lastly, Leishmania promotes its survival within the macrophage by down regulating MHC molecules and by preventing their antigen presentation. The glycosylinositolphospholipid Leishmania component has been found to prevent the expression of both classes of MHC molecules. The degree of gene suppression correlates with both the duration of infection and the parasitic load (17). This is supported by the fact that increasing quantities of IFN-g do not remedy this. (Recall that IFN-g stimulates infected macrophages to die.) For the few MHC molecules that are produced, however, the parasite is able to prevent peptide loading. Gp63 cleaves the CD4+ molecules on T cells, undermining the interaction between antigen-presenting cells and T helper cells (18). One hypothesized mechanism involves the release of proteolytic enzymes by the parasite in the vicinity of peptide loading onto the MHC molecules, essentially degrading the peptides "on the spot" (19). Thus, Leishmania is capable of subverting key macrophage accessory functions that are required for the induction of T cell-dependent anti-parasite immunity.

Streptococcus Pneumoniae
Streptococcus pneumoniae is a human pathogen that frequently causes pneumonia, otitis media, septicemia, and meningitis (47). The mucosal epithelium of the nasopharynx is the primary site of

colonization, and individuals can carry up to four different serotypes asymptomatically (63). In some cases, perhaps in conjunction with a viral infection, the host is predisposed to symptomatic pneumococcal infections including sinusitis, otitis media, and pneumonia. In rare cases, sepsis develops and seeds infections at distant sites (e.g., meningitis). Recent studies of the natural course of disease progression have suggested that pneumococcal adherence to mucosal surfaces involves cytokine-mediated upregulation of platelet-activating factor receptor (63). However, there remain considerable gaps in our understanding of the mechanism of pneumococcal invasion of host tissue. Identification of the molecules important in the disease progression has become easier with the development of mouse models of pneumococcal diseases that replicate nasopharyngeal colonization that can lead to pneumonia and sepsis (67). Host defense against S. pneumoniae involves mainly acute-phase responses as well as antibodies to pneumococcal antigens. C-reactive protein is an acute-phase protein that binds to phosphocholine moieties within cell wall polysaccharide (C-PS) in the presence of calcium (65). C-reactive protein promotes phagocytosis of S. pneumoniae by human leukocytes (34) and protects mice against fatal pneumococcal infection (60, 71). Either short-term or chronic ablation of tumor necrosis factor and tumor necrosis factor receptor (TNF/TNFR) function renders mice more susceptible to S. pneumoniae, suggesting that cytokine-mediated inflammatory processes play an integral role in host defense against pneumococcal infection (50, 61). Classic studies demonstrated that antibodies to capsular polysaccharide (Caps-PS) are an important component of the adaptive immune response to pneumococcal infection (38). Subsequent studies, however, revealed that antibodies to C-PS (41), phosphocholine (5), or pneumococcal proteins (e.g., PspA) (7) can protect mice against pneumococcal infection. Thus, antibodies to many different pneumococcal antigens could be important in host defense. CD40L is critical for humoral responses to T-dependent antigens, whereas antibody responses to type II T-independent antigens (e.g., TNP-Ficoll) occur independently of CD40L (22, 23, 25, 33, 51, 69). CD40L also regulates fibroblast (11, 70, 73), epithelial (72), and endothelial (17, 30, 46, 54) cell function through regulation of adhesion molecule expression and production of prostaglandins, cytokines, and chemokines. The pivotal role of CD40L in protection from viral infections (4, 42, 52, 62) and from several, but not all, intracellular pathogens or parasites is well established (10, 14, 16, 26, 29, 32, 36, 59,74). However, the role of CD40L in protection from infection with extracellular bacteria is less well understood. A protective humoral response to Borrelia burgdorferi, the causative agent of Lyme disease, is elicited independently of CD40L (21). In a recent study, Wu et al. demonstrated that the antiphosphocholine-specific immunoglobulin G (IgG) responses elicited by immunization with a nonencapsulated, nonvirulent variant of S. pneumoniae are impaired in T-cell-deficient or CD40L(/)mice (68). The present study was undertaken to determine whether CD40L is essential for protection from an encapsulated strain of S. pneumoniae. The effect of anti-CD40L (MR-1) on humoral responses to pneumococcal antigens as well as susceptibility to pneumococcal infection was examined.