OPTIMIZACIÓN USANDO EL PROGRAMA EES

Gonorrhea is another example for a STD and is caused by Neisseria gonorrheae, a gram-negative diplococcus. It is an exclusive human pathogen that primarily infects the urogenital epithelia but can also lead to infection of other EC surfaces.

Gonococcal infection is with over 60 million cases a major global health problem with highest incidences in less developed countries (444). However, more than 300,000 cases are reported to the Centers for Disease Control and Prevention in the United States each year but an estimated 600,000 people are infected (197).

Gonococcal infections usually remain localized to the sites of primary infection, the male urethra and the female cervix. Gonococcal cervicitis often remains asymptomatic but subsequently tends to spread to the upper genital tract inducing chronic complications (353). Ascending infection occurs in up to 45% of infected women and can result in pelvic inflammatory disease (PID) which can cause infertility and ectopic pregnancies due to fallopian tube blockage (1330). Repeated infections are rather common, which suggests antigenic variations in the organisms or an ineffective immune respose to infection.

Furthermore, the gonococcus-induced increase in the local expression of viral RNA together with the loss of mucosal integrity due to an acute inflammatory response is associated with increased susceptibility to HIV-1 infection (211). Despite the presence of effective antibiotic treatment these health concerns demand the need for further investigation on the mechanisms of the infection in order to develop a vaccine against gonorrhea.

(1) Mechanisms of N. gonorrheae to evade the immune response

Virulence factors

Only recently it has been shown that N. gonorrheae can invade mucosal ECs and are intracellular during human infection (39).

A repertoire of virulence factors have been identified and allow this bacterium to successfully adapt to variable microenvironments within its sole human host and are thought to play a role in EC invasion. These virulence factors are responsible for gonococcal evasion of the human immune system and explain the tendency to chronificate. These factors consist of outer membrane constituents such as porin, pili, opacity-associated outer membrane proteins (Opa), reduction modifiable protein (Rmp) and lipooligosaccharides (LOS) (353).

Pili seem to modulate host cell signaling mechanisms to aid gonococcal epithelial invasion and participate in forming an initial attachment with host cells (1372). They may also provide a mechanism by which nonmotile gonococci are able to colonize and to ascend mucosal surfaces through their ability to exhibit twitching motility (1469). The transmembrane protein CD46 involved in regulation of complement activation has been demonstrated to serve as a receptor for gonococcal pilus in human cells (653).

Opa proteins are thought to contribute to the cellular trophisms exhibited by gonococci and are divided into two classes based on their ability to differentially recognize host cell surface molecules (353). Opa50 recognize host cell heparin sulphate proteoglycans and Opa52 recognize carcinoembryonic antigen-related family of cell adhesion molecules (CEACAM or CD66).

LOS play a role in attachment of the pathogen to epithelial tissues and is a target for bactericidal antibodies found in normal human serum (160). Variations of LOS

between strains which results in constant antigenic variation. This spontaneous conversion of oligosaccharide determinants can change the manner in which the gonococcus associates with host tissues and, hence, can potentially alter the course of gonococcal disease.

Porin, a water-filled channel through which small molecules traverse the gonococcal outer membrane, is thought to play multiple roles in potentiating disease caused by N. gonorrhoeae. Porin molecules trigger variable functional responses within host cells depending upon the particular porin and the host cell type under study. A unique feature of gonococcal porin is its ability to translocate into eukaryotic cell membranes and form a voltage-gated channel (1484).

By demonstrating the ability of porin to induce apoptosis in ECs (985) it is proposed that porin plays a role in the cytotoxicity observed in fallopian tube organ culture and in the shedding of ECs which occurs in vivo during mucosal infection (353). In contrast, gonococcal infection of primary human male urethral ECs results in antiapoptotic events (116). It is hypothesized that the enhanced survival of the urethral EC may allow the bacterium to proliferate within an intracellular, protective environment and, consequently, promote gonococcal colonization. Porin may also facilitate the cytoskeletal rearrangements required for actin-mediated entry of the gonococcus into its target host cell (1493).

These virulence factors together with the high heterogeneity and adaptability with repeated phase and antigenic change may be a way to downregulate the functional immune response of the host. Pathogens use at least two basic strategies to survive the host`s immune response. Some avoid provoking specific host defenses and others induce immune responses but possess the ability to evade the consequences. The high rate of reinfection despite the presence of antigonococcal antibodies leads to the assumption that N. gonorrhoeae evades the host’s immune response.

Indeed, N. gonorrhoeae possess several mechanisms which could potentially thwart the effects of immune responses directed toward this organism in vivo, including hypervariation of surface antigens, resistance to complement- mediated bacteriolysis, and the production of IgA1 protease (530).

Resistance to complement-mediated bacteriolysis

Gonococci possess several potential mechanisms to avoid complement-mediated bacteriolysis, including sialylation of LOS, induction of blocking antibody to Rmp and binding of complement downregulating proteins such as C4bp to particular

porin domains (1229). However, it seems unlikely that complement-mediated, IgG- dependent bacteriolysis operates at mucosal surfaces, where a fully functional complement system is not usually present (1396) and IgA may interfere with complement activation. Both IgA and IgG in genital secretions have been shown to inhibit adherence of gonococci to ECs (1428).

IgA1 protease

Vaginal washes from patients infected with N. gonorrhoeae have been found to contain IgA1 protease activity demonstrable in vitro and were able to cleave exogenous IgA1 in a manner suggestive of IgA1 protease activity (120). However, despite the presence of substrate, and contrary to expectations, Hedges et al. did not detect any evidence of IgA1 cleavage fragments by gonococcal IgA1 protease in cervical mucus or vaginal wash samples (530). Nevertheless, all of the clinical isolates of N. gonorrhoeae infecting these patients produced IgA1 protease in vitro.

These apparently contradictory results may be explained by two linked hypotheses. First, N. gonorrhoeae may not be present in the lumen or on the mucosal surface in sufficiently high numbers but rather may colonize a subepithelial niche (530). This hypothesis is supported by the lack of significant local immune or cytokine responses in women infected with N. gonorrhoeae. Therefore, the lack of detectable IgA1 protease activity in cervical mucus, in addition to the lack of local host responses, may simply be due to small numbers of bacteria at that site.

Secondly, N. gonorrhoeae may require IgA1 protease for survival within as well as outside the host tissues.

In addition, there is recent evidence that the lysosomal-phagosomal protein LAMP-1 on ECs is cleaved by neisserial IgA1 protease (820). Growth of gonococci within ECs was enhanced by cleavage of LAMP-1 which may contribute to intracellular gonococcal survival and facilitate escape from antibodies and complement.

Differences of gonococcal infection in male and female

Interestingly, there are important differences between gonococcal genital infection in men and women and N. gonorrhoeae has evolved variable pathogenic mechanisms to ensure its survival in the distinctly different microenvironments found within the male and female (uro)genital tracts (353).

In male urethral epithelium, the interaction of gonococcal LOS with the asialoglycoprotein receptor present on the urethral EC mediates invasion and

Cytokine release contributes to the usually symptomatic nature of gonococcal disease in men and is accompanied by a large influx of PMNs, which in turn contribute to the observed cytokine release and inflammation. The interaction of gonococci with PMNs is mediated by the interaction of Opa gonococcal proteins and CEACAM host cell proteins (1192). The specific interactions occurring between these two families of proteins may dictate specific host cellular responses and the survival or death of phagocytosed gonococci. The PMN response to gonococci is further modulated by gonococcal porin, which inhibits PMN degranulation and the production and release of toxic oxidants from the host cell to the extracellular milieu. Transmission of gonococci to a sexual partner is then partly aided by the ability of gonococci to bind to human sperm which also were found to express asialoglycoprotein receptor (515).

In contrast to the inflammatory response generated predominately with gonococcal infection of the male urethra, 50 to 80% of women with lower genital tract N.

gonorrhoeae infection are asymptomatic, and 70-90% of women with disseminated

infection lack signs of genital tract involvement (1330).

The clinical findings that there is neither an antibody response nor elevated local cytokine levels in women with gonococcal infection are consistent with the ability of the gonococcus to evade and subvert host immune function (531).

Cervical epithelia provide a source of alternative pathway complement activity, yet, at a level comparable to only approximately 10% of that observed for human serum (1440).

Within minutes of infection of primary cervical ECs, complement protein C3b is deposited on the lipid A portion of gonococcal LOS and is rapidly inactivated to iC3b (354). This is supported by the predominance of iC3b in comparison to C3b on the surface of clinically isolated gonococci (1460).

Analysis of clinical biopsies obtained from women with culture-documented gonococcal cervicitis and infection studies performed with primary human cervical ECs indicate that complement receptor 3 (CR3) on female genital epithelia serves as the primary receptor for N. gonorrhoeae adherence to and invasion of the ectocervix and endocervix (355).

Binding of gonococcal pilus to the I domain of CR3 probably allows the gonococcus to overcome the electrostatic repulsion between its own cell surface and that of the cervical cell and may juxtapose the gonococcus at the cervical cell surface, where complement concentrations would be expected to allow efficient opsonization for the subsequent intimate adherence of iC3b, i.e. converted C3b, and gonococcal porin to

the I-domain. Binding of the gonococcus to CR3 requires the cooperative action of iC3b bound to the gonococcal surface in conjunction with gonococcal porin and pilus (355). This ligand binding to the I-domain of CR3, however, does not invoke a proinflammatory response in professional phagocytic cells and cellular fate of gonococci is not clear (353).

Interestingly, menses is associated with an increased risk to women for PID and for disseminated infection (1373). C3 production by the cervical epithelium exhibits cyclic variability, and the highest levels of C3 are detected during menses (521). Additionally, a correlation can be made between the presence or the absence of Opa and the site of gonococcal infection. Opa- gonococci are predominate within the fallopian tubes and in the cervix at the time of menses while Opa+ gonococci are found predominately within the male urethra and within the cervix at the time of ovulation (353).

Ascent to the upper female genital tract might be facilitated through the ability of gonococci to exhibit twitching motility, in conjunction with hormonal changes which influence the mucosal epithelium and the expression of complement and molecules serving as gonococcal receptors within the female genital tract (353). Microscopic analysis of tissue biopsies indicates that the expression of CR3 progressively decreases in an ascending manner from the ectocervix to the fallopian tubes (354). But expression of the lutropin receptor (LHr) which might serve as gonococcal receptor in upper genital tract epithelia (1331) increases in an ascending manner to fallopian tubes with highest levels at menses (521). The presence of LHr on the human uterus, placenta, decidua, and fetal membranes may partly contribute to the fact that the increased risk of spontaneous abortion associated with N. gonorrhoeae infection is due to a gonococcus-LHr interaction occurring on deciduas and placental membranes (1190).

(2) Immune responses to gonococcal infection

Igs

Early studies using enzyme immunoassay determined the presence of IgM, IgA and IgG antibodies to gonocoocal pili antigens in serum (930). In all Ig classes, a significantly higher mean antibody activity and a higher percentage of positive sera were found in men and women with N. gonorrhoea than in controls. The magnitude of antibody response was higher among infected women than men, especially in the IgM class. Another study found serum and local IgG and IgA to be produced against

several antigens during goncoccal infection, although the quantity of antibody was greater in serum (601).

As mentioned earlier, recent preliminary evidence indicated, however, that while antigonococcal antibodies were detected in infected patients, the levels of both serum and antigonococcal antibodies in genital secretions were surprisingly low (532).

A following study measured the concentrations of total IgA1, IgA2, IgG, and IgM in cervical mucus, vaginal wash, and serum samples from volunteers without demonstrable infection, from volunteers in whom only N. gonorrhoeae was detected, and from volunteers infected with other pathogens (C. trachomatis or T. vaginalis) with or without N. gonorrhoeae (531).

There were no differences between the concentrations of total IgA1, IgA2, IgG, and IgM in genital tract secretions in patients with different STDs compared with non- infected women. Moreover, levels of IgA1, IgA2, IgG, and IgM antibodies specific for

N. gonorrhoeae MS11, a widely studied gonococcal strain, in female mucosal

secretions and serum were found at low levels in both uninfected and infected women. IgA1 antibody levels in serum, but not in secretions, were higher in female patients infected with N. gonorrhoeae than in noninfected patients while the levels of IgG and IgM antibodies in serum and secretions were not different between gonococcus-infected and noninfected patients (531).

A history of previous infections with N. gonorrhoeae did not alter the antibody levels in patients with a current infection except for arising levels of serum IgA1 antibody. These results further support the possibility that repeated infections with N.

gonorrhoeae are common because there is little development of immune memory

and therefore only minimal levels of protective immunity.

One potential explanation for the paucity of antibody responses to N. gonorrhoeae in uncomplicated genital tract infections may be related to the absence of organized mucosa-associated lymphoid tissue, such as the Peyer`s patches of the small intestine, which are recognized as major sites for the uptake and processing of antigens leading to generalized disseminated mucosal immune responses as described earlier.

Although in some studies local vaginal antibody responses were recorded, intravaginal immunization in humans appeared to be inefficient in inducing either circulating or generalized mucosal antibody responses compared to oral or nasal immunization (743). In contrast to the genital tract, the rectum contains lymphoid

follicles resembling Peyer`s patches that likely serve as an inductive site of the common mucosal immune system. In addition, it has been suggested that these sites may preferentially supply specific antibody-secreting cell precursors to the adjacent genital tract which shares the same lymphoid drainage (272). Therefore, it seemed likely that persons infected at both the rectum and genital sites might be expected to display enhanced antibody responses to the infecting organism, both in the genital tract and perhaps also in remote secretions.

Due to the prevalence of rectal infections with N. gonorrhoeae in the mentioned study it could be examined whether more pronounced antigonococcal antibody responses were generated by gonococcal infection at a site known to contain organized inductive lymphoid tissue.

There was a small effect of rectal infection on the levels of isolate specific IgG in cervical mucus but overall only little difference in antibody levels in patients with cervical compared with cervical and rectal infections was found, suggesting that rectal infection was no more efficient than the genital tract infection for inducing humoral responses to N. gonorrhoeae (531).

A recent study by Pantelic et al. demonstrated that N. gonorrheae via their Opa protein has the ability to suppress antibody production by killing CEACAM1- expressing B cells (1075).

Cytokines

This relative paucity of antibody response provokes the question as wether there is a cytokine response to gonococcal infection as it is seen in female urinary tract infection and experimentally infected men (1166).

Therefore, the levels of IL-1, IL-6, IL-8, IL-10, and TGF-β in sera and genital tract secretions from women with gonococcal cervicitis and other genital infections were examined (532). Surprisingly, the local levels of all these cytokines in genital secretions were not elevated in women with gonococcal cervicitis compared with levels of uninfected persons. In contrast, serum IL-6 levels, but not IL-8 and IL-1 levels were significantly elevated in gonococcus-infected women.

Serum, but not local, IL-1 and IL-6 levels were elevated in patients concomitantly infected with Trichomonas vaginalis or Chlamydia trachomatis in addition to N. gonorrhoeae compared with levels in patients infected with any single organism (532).

ectocervical epithelia (396). Experiments with whole gonococcal lysates revealed that the IL-8 and IL-6 response by cervical and vaginal ECs was not restricted to the interactions with viable gonococci.

Similarly, a recent study has shown that viable N. gonorrhoeae is not essential for proinflammatory response by innate immune cells, since mature human macrophages generate an array of cytokines and chemokines in response to purified gonococcal surface antigens (861).

These findings suggest that gonococcal components can stimulate proinflammatory responses, which are independent of either gonococcal metabolic activity i.e. viability or entry into the host cells.

T lymphocytes

Gonorrhea typically correlates with a transient reduction in T cell counts in blood,

and these populations recover when gonococcal infection is resolved. Opa proteins have been shown to bind CEACAM1 expressed by primary CD4+ T cells and suppress their activtion and proliferation (134).

On the basis of the absence of cytokines and the low levels of antigonococcal antibody detected during uncomplicated cervical infections by N. gonorrhoeae it is proposed that in addition to their ability to evade the consequences of immune responses, gonococci either fail to induce, or possibly actively suppress, the host`s immune and inflammatory responses.

(3) Development of vaccines

As N. gonorrheae is an obligate human pathogen, the development of a vaccine has

been hampered by the unavailability of a convenient and simple animal model. Attempts to infect or colonize the genital tracts of different animal species have been unsuccessful (49).

Certain vaccines have been evaluated in human males but earlier prototype gonococcal vaccines have shown limited or no protection against reinfection with N.

gonorrhoeae despite the generation of serum antibody responses against the vaccine

antigens. A trial with gonococcal pilus vaccine, which has been shown to be safe as well as antigenic and resulted in the production of specific antibodies, failed to protect men (1429, 132).

A second vaccine consisted of porin protein as a systemic immune response to