The Kiss of Death

by Joseph William Bastien

  Protective and Nonprotective Antibodies

  During Chagas’ disease, both protective and nonprotective antibodies are produced. Lytic antibodies (LA) recognize epitopes (specific markers) on the surface of living trypanosomes, attach to these epitopes, and initiate the complement cascade, which includes enzymes that lyse the parasite. Among chronically ill patients, there are varying levels of lytic antibodies. Those patients that show the least clinical manifestations have the greatest number of lytic antibodies that react with epitopes exposed on the surface of living trypanosomes. This explains why acquired immunity from severe symptoms of the acute phase requires the continual presence of living trypanosomes.

  The nonprotective antibodies are called CSA (conventional serological antibodies) and do not recognize epitopes of T. cruzi. Patients with the most dramatic clinical manifestations have the greatest number of CSA, which are not inert antibodies but contribute to pathogenesis. Experiments have been performed giving people ground-up antigens of T. cruzi. These antigens from nonliving T. cruzi organisms induced nonspecific T- and B-cell polyclonal activation and thereby vast amounts of ineffective antibodies that cause more damage to hosts than parasites, similar to what happens to the body in African sleeping sickness and malaria infections.

  In this experiment, as well as among chronic patients with severe clinical manifestations, the immune system is being inundated with many different T. cruzi antigens, a few of which are vulnerable targets but the vast majority of which are no more than smoke screens. A person’s humoral immune system produces high levels of anti-trypanosomal antibodies, but these antibodies are not targeted against epitopes on the surface of living parasites. Rather, these CSA are targeted against antigens that are not important to the parasite, or are unable to be reached, so the CSA are unable to lyse the organism. In popular terms, T. cruzi antigens stimulate polyclonal activation, which is like throwing in a large group of antigens and confusing the system, which then overreacts with too many ineffective punches that do more damage to living cells than to parasites by causing inflammation, fever, and lesions.

  The fact that Chagas’ disease patients vary greatly in their proportions of LA and CSA antibodies partially explains the wide range of clinical manifestations exhibited. As already discussed, acute patients with severe symptoms have high levels of CSA and low levels of LA; conversely, those with high levels of LA frequently do not suffer symptoms of the acute phase. This may explain why only about one-third of infected patients suffer the acute phase. The key variable is how each individual immune system deals with specific surface epitopes of antigens that are important in acting against the parasite. Another factor contributing to the LA/CSA response may be the strain of the parasite; different strains sometimes elicit sharply different antibody responses.

  Because low levels of LA correlate with clinical manifestations and because individuals vary in the amount of LA produced, it is important to determine the presence of lytic antibodies in infected patients. ELISA, complement fixation, indirect agglutination tests, and radio immunoassays are not specific enough to distinguish which particular antibody is able to kill T. cruzi. One serological test is complement-mediated lysis, in which the patient’s serum is bound to trypomastigotes in vitro and exposed to complement. If the complement lyses the parasites, then LA is present in the blood. Indirect immunofluorescence with living trypanosomes is another assay that can determine the protective effect of serum: technicians study antibodies fixed to the surface of living trypanosomes with immunofluorescence to see if they are interacting with epitopes on the surface of the parasite. A third method is serum neutralization, in which living trypanosomes are exposed to a patient’s serum. If the parasite is neutralized, then the serum contains lytic antibodies.

  These tests are important to determine a patient’s progress against T. cruzi infection and whether chemotherapy should be used. Patients with low LA levels will not be helped as much by chemotherapy as those whose antibodies are assisting in lysing the parasite. Moreover, as patients are being treated and being cured, LA levels decline rapidly, giving an indication of effective treatment.

  This also explains why an active infection is required to stimulate protective immunity: cured patients no longer have LA in their systems. LA are manipulated by parasites so that they are produced at certain times and released under certain circumstances. Parasites regulate their number and degrees of infection in order that overpopulation does not become a problem for their survival and that their hosts remain alive until they can complete their own life cycles.

  Also influencing the severity of symptoms, strains and stages of T. cruzi vary in their susceptibility to complement-mediated lysis. Some strains are lysed more effectively by human complement than are other strains, and the virulence of strains conversely may be related to the parasites’ susceptibility to complement. Regarding T. cruzi stages, blood-form trypomastigotes are more susceptible than are metacyclic trypomastigotes and intracellular amastigotes. Metacyclic trypomastigotes are initially exposed to complement, and in chickens these parasites are lysed. Metacyclic trypomastigotes quickly go intracellullar, especially into macrophages, where they transform and multiply into amastigote forms. Because of their intracellular location, amastigotes resist complement-mediated lysis. After several generations, amastigotes transform into trypomastigotes that circulate in the human blood. Not many trypomastigotes are released into the blood; many of them go into cells. These trypomastigotes show a wide variation in their resistance to complement-mediated lysis, but they are much more susceptible to complement-mediated lysis in the presence of specific T. cruzi antibodies than are other forms.

  Evasive Strategies

  Intracellular hiding is a very important evasive strategy of T. cruzi, because the blood is the primary battleground of the humoral immune response. If T. cruzi trypomastigotes remained in the blood for a month, this would give the T and B lymphocytes sufficient time to activate very specific IgG immunoglobulins against them. During the acute phase, polyclonal stimulation occurs and parasites are chased into cells. After polyclonal activation subsides, immunosuppression follows, which provides more time for trypomastigotes to enter cells.

  Some lesser strategies are evident. Trypomastigotes show rapid surface turnover, capping, shedding, and some interstrain antigenic variation of their coats, but not to the degree exhibited by the T. brucei-complex of organisms. If antibodies attach to trypomastigotes, trypomastigotes can shed them if they act quickly enough.

  Trypomastigotes are also able to evade host antibodies by binding to the Fc (crystallizable fragment) portion and by fabulation. T. cruzi have a surface component that enables their antigens to bind to the Fc end and not to the FAB (antigen-binding fragment) end of the immunoglobulin molecule. If antigens bind to the Fc end, then this end is blocked from activating complement, which is necessary to lyse the parasite. This is especially true for antibodies directed specifically at nonsurface epitopes, which they cannot reach, since antigens on the trypomastigote’s surface bind to the Fc portion of IgG. Antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent complement lysis are both dependent on the Fc portion being exposed. Trypomastigotes become coated with host antibodies clinging to antigens on their surfaces, but with their FAB extended and nonfunctional.

  Fabulation is when trypomastigote antigens bind to the FAB end of the molecule and cleave off the Fc portion, which literally disables the antibody. Trypomastigotes have surface proteases (protein-eating enzymes) that cleave off the Fc end of the antibody. Without its Fc end, the antibody is left with an ineffective little piece of protein dangling from its surface that is unattractive to complement or to cells.

  Although it is not surprising that T. cruzi has all these mechanisms for evading the host immune system, African trypanosomes rely most heavily on antigenic variation. African trypanosomes don’t have the need to use anything but antigen variation, which is enough to enable them to avoid the destruction of all the
ir number by host immunity measures. A parasite may have four or five mechanisms for evasion, and then another one appears that is better than the others, but the parasite doesn’t then get rid of the less-effective ways, at least not for many, many generations. These mechanisms will be selected in those organisms that don’t have the better one. T. cruzi, then, has not attained such a refined mechanism of evasion as antigenic variation, and so it utilizes more than one evasive mechanism. All of which work toward the same end: the survival of the species.

  Autoimmune Components of Pathogenesis

  The severe symptoms of Chagas’ disease come in large part from autoimmunity responses (see Andrade 1994, Brener 1994, Iosa 1994). Thus, most of the pathology associated with T. cruzi infection is referred to as immunopathology. Throughout the chronic phase, a tremendous inflammatory process accompanies cells invaded by T. cruzi amastigotes, especially the amastigotes that live for a long period of time. This inflammatory process most frequently focuses in the heart, causing chronic myocarditis, but it also occurs within the esophagus and colon. This inflammatory response is directed against amastigotes that, over a period of time, slowly release themselves from the cells that they have invaded. This begins an inflammatory process against the escaped amastigotes and destroyed cells left behind. Monocyte or mononuclear infiltrates focus within specific locations where the parasites emerge and eventually invade other tissues, including the heart and neuron tissue. These infiltrates also enter myocardial plexes and plexes that serve the digestive tract. Plexes are nerve nets that serve various organs. Neuronal degeneration can occur during the acute phase in those who are experiencing severe attacks, especially children under the age of five, but this degeneration most frequently occurs during the chronic phase of the infection.

  During the acute phases, polyclonal activation incites T and B cells, which produce antibodies not directed against the parasite in ways that will protect the host but rather against epitopes the parasite possesses and may share with host cells. These CSAs or nonproductive antibodies damage heart and nerve cells, frequently leading to death.

  During the chronic phase, T. cruzi protects itself by mimicking host antigens which are shared with heart and nerve cells. Molecular mimicry between T. cruzi and host nervous tissue is the most current and acceptable theory of how the immune system is responsible for tissue lesions on infected organs (see Avila 1994 and PAHO 1994 for a discussion of this theory). T. cruzi have on their surfaces various host plasma proteins and immunoglobulins (antibodies), as well as shared heart- and nerve-cell antigens. During the chronic phase, damage of the cardiac system leads to conduction abnormalities, which can be detected through the measurement of cardiac function. Gastrointestinal damage begins with the reduction of muscle tone and ends with muscular atrophy of the smooth muscles of the gut, esophagus, and colon so that these organs dilate and cannot contract to pass food through, resulting in blockage. Patients suffer chronic dysphagia (difficulty in swallowing) and constipation, which can include an enormously enlarged colon (megacolon) and esophagus (megaesophagus).

  These severe clinical manifestations, however, cannot adequately be explained by inflammatory responses of the immune system to a few parasites being attacked with a focal inflammation. During the chronic phase, T. cruzi reproduces in numbers, and the immune system attempts to keep them in check; but, at some point, the inflammation process rapidly spreads out from focal areas where parasites are emerging, to attack human cells. This diffusion heralds the critical point for chronically infected patients.

  This is not just a matter of parasite antigens attaching to self cells and rendering them nonself cells so that human antibodies mistake human cells for parasite cells, but rather it is a situation where human antibodies attack self cells simply for what they possess. T. cruzi immunizes people to their own antigens: it causes the human immune system to create antibodies that target antigens belonging to cardiac and neuronal cells and which then lyse them with complement. The immune response attacks self cells in two ways: parasites frequently alter the normal structure of host cells or attach parasite antigens to these cells; or, using an even more effective method, these parasites can get the immune system to think that it is attacking the parasite while it is in fact attacking itself. T. cruzi can do this because it has surface molecules that mimic those on the surface of host cells.

  This has been demonstrated in experiments with rabbits. When lymphocytes from chronic chagasic rabbits were injected into healthy rabbits, these lymphocytes bound to and destroyed heart cells. This provided evidence that a cell-mediated autoimmune response is involved in the generation of cardiomyopathy and other pathogenic events that occur with the disease. If spleen cells from T. cruzi-infected rabbits are placed in vitro with normal rabbit heart cells, the heart cells are killed. Spleen cells from the infected rabbit contain both lymphocytes and monocytes sensitized to the parasite antigen.

  Further proof is found in the fact that serum from chagasic patients invariably contains autoantibodies that attack heart and neuronal cells in vitro. These autoantibodies attach and bind to cardiac epithelium and interstitial tissue, blood vessels, and muscle tissue, and are technically referred to as EVI antibodies (endothelial, vessel, and interstitial). EVI antibodies are detected at high levels in the serum of chronically ill chagasic patients and at extremely high levels in the serum of those with cardiac abnormalities.

  The question is, how does T cruzi make human antigens look like T. cruzi antigens? When the host immune system attacks T. cruzi, it recognizes many epitopes on the parasite’s surface that are nonself and belong to the parasite. The surface of T. cruzi has many molecules on its surface which have epitopes that allow macrophages to attach to the surface and phagocytize the parasite. Macrophages pick up epitopes unique to T. cruzi as well as others the parasite shares with the host; so, when T cells develop and receive this information, they confuse parasite and human epitopes. Human cells can also be rendered nonself by attaching to them something that is nonself, such as a parasite antigen. The human immune system then attacks its own cells. When the immune system begins to attack its own cells, it also processes information about this system of antigens and includes all those that are related to it macromolecularly. It is clueing-in on some antigens that are not nonself and develops a response against its own cells. Therefore, when the immune system goes after T. cruzi, which has on its surface some antigens that are similar to those of the host, the immune system picks up epitopes of self cells that have been disarranged because they are in close association with these nonself antigens and produces antibodies against the human cells. The immune system response is not specific enough to determine which molecules belong to the parasite and which to itself.

  The antigenic epitope involved in this autoimmune reaction against cardiac cells contains laminin (a basement membrane glycoprotein). Tested sera from infected humans and monkeys contained high levels of EVI antibodies that reacted with laminin. EVI antibodies are anti-laminin antibodies. This was detected by pouring serum down a sepharose (gel) column with laminin bound to it so that only anti-laminin antibodies would stick to it. This affinity chromatography test is called a laminin sepharose affinity column. Laminin is a very important glycoprotein on the surface of many cells, especially those of the heart. If rabbits are immunized with laminin, they produce antibodies that cross-react with T. cruzi and EVI cells. Conclusions are that laminin may be the major antigenic component inducing EVI antibodies and that laminin is found on the surface of T. cruzi and cardiac cells.

  However well laminin explains myocardiopathy, it is not found in large amounts on the surfaces of neuronal tissue, so there must be another cross-reacting antibody to explain the degeneration of neuronal tissue and muscles of the gut.

  In conclusion, T. cruzi is a complex organism that has elaborate mechanisms for survival in its host. Within its millions of years of existence, T. cruzi has evolved a number of strategies for evading and manipulating mechanisms of the human immu
ne response. In an elaborate chess game of moves and countermoves, T. cruzi has literally checked the king, although the game is not over, as scientists rapidly are uncovering complexities of the relationship between T. cruzi and the vertebrate immune system. The second theme implicates the human immune system for sometimes doing more damage than good: the fact that a protozoan such as T. cruzi can turn this system around so that it attacks itself is a strategy any warrior could learn from, but the most important lesson from T. cruzi is that it provides us with information and the stimulus to do more research on this process. Parasitology and immunology are extremely complex disciplines needing more study, and Chagas’ disease has proven to be an important and interesting model in both areas of study.

  APPENDIX 12

  Diagnostic Tests

  In 1909, when Carlos Chagas took a sample of the child Rita’s blood and examined it under the microscope, he saw trypanosomes. He was able to find them because they were abundantly present at this time, although, generally speaking, it is more difficult to find them in a blood sample. Trypanosomes of T. cruzi can be found in patients during the acute phase of Chagas’ disease by direct examination of the blood and later by centrifugation of clotted blood, xenodiagnosis, and animal inoculation. One problem is the possible misidentification of parasites in examination of the bloodfor example, the spirochete of syphilis has been confused with T. cruzi. If T. cruzi is reported in blood samples, retesting is recommended. Tables 3 and 4 concisely provide a list of the different tests and their validity.