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Chlamydia and tubal subfertility: Australia's fertility at risk.

Presented at the NHAA 7th International Conference on Herbal Medicine, July 2010, Coolangatta Australia

Introduction

Chlamydia trachomatis (chlamydia) is the most commonly diagnosed bacterial sexually transmitted infection (STI) in Australia (Hocking 2008). It is acquired during oral, vaginal or anal sexual contact with an infected partner (Aust Govt Dept Health & Aging 2005). It is difficult to estimate the number of cases of chlamydia because of the large number of potentially asymptomatic persons who would be identified only through screening, which is not available nationally (Hocking 2008). Despite this, notification rates have risen dramatically, from 47.4 per 100 000 in 1997 to 203 per 100 000 in 2005 (Aust Govt Dept Health & Aging 2005). In 2001 there were 11 859 notifications in Australia for young people aged 12-24 years (Aust Govt Dept Health & Aging 2005). Of these 69% were for females, 2% were aged 12-14 years, 17% for those aged 15-17 years and 82% for those aged 18-24 years (Aust Govt Dept Health & Aging 2005). Other reports from Western countries are that the prevalence of chlamydia amongst young women aged 15-24 years has been reported as being 3.4-17.6% (Land 2002).

Organism

Chlamydia is an obligate intracellular pathogen that resides within a specialised vacuole and has a biphasic developmental cycle (Friss 1972). Chlamydiae have a slow developmental cycle, which explains the insidious onset of chlamydial infection (Mgiga 2006). There are two forms of the organism. The elementary body (EB) is the infectious form (Mgiga 2006). Once the EB attaches to the columnar epithelial cell it is incorporated into the cell by a process of pinocytosis, in which the organism is surrounded with a phagosome membrane (Friss 1972). Chlamydial particles remain within the phagosome membrane during their entire existence within the host cell (Eschenbach 2010). This membrane isolates the organism from the cellular immune system protecting it from recognition and attack by usual cellular defence mechanisms (Eschenbach 2010). After being present for a few hours within the cell, the EB is transformed into a non infectious reticular body (RB) (Mgiga 2006). Protected by the phagosome membrane from destruction, the RB directs virtually all of the cellular produced adenosine triphosphate for its own growth (Eschenbach 2010). In this stage the organism becomes metabolically active and it begins to actively synthesise protein (Mgiga 2006). The RBs grow and eventually divide by binary fission producing new infectious EBs.

The multiplication process repeats itself until the chlamydial inclusion body becomes the dominant structure in the host cell (Armstrong 1963). Numerous new infectious EBs are released back into the extracellular environment when the inclusion body ruptures, and the EBs infect adjacent epithelium while the host cell is destroyed (Mgiga 2006).

The 24 hour replication process required for chlamydiae is rather slow compared with the 2 to 4 hour division time of most classic bacteria (Eschenbach 2010). Therefore it is not surprising to find that there characteristically is a long latency period between the time chlamydiae are acquired and symptoms are produced (Eschenbach 2010).

The major problem of control of genital tract chlamydia infections is that as many as 80% of women and up to 50% of men are asymptomatic (Land 2002). This results in a large reservoir of undiagnosed infected individuals who can transmit the infection to sexual partners (Land 2002). This 'silence' promotes bacterial progression, even to the most internal tissues where upper genital tract infections and late complications may develop.

Clinical spectrum of chlamydial pelvic inflammatory disease (PID)

Both symptomatic and asymptomatic chlamydial infections can damage the reproductive tract. The major sequelae of chlamydia infection in women is ascending infection leading to PID, ectopic pregnancy and infertility (Barrett 2005). The sequelae of PID that are responsible for infertility include chronic interstitial salpingitis, hydrosalpinx, salpingitis isthmica nodosa, and periadnexal adhesions. Infertility may also occur because of abnormal secretory, ciliary and peristaltic function of the fallopian tube and/or immune rejection of the embryo (Novy 2008).

Evidence does exist that in about half of the asymptomatic women with lower genital tract infections the microorganisms will be cleared spontaneously. In the remaining group cervical infections may persist over many years, and in only a small proportion (about 10%) cervical infections will ascend to the upper genital tract and induce pelvic inflammatory disease (Land 2002).

Women with asymptomatic chlamydia infections are less likely to seek medical attention than women with genital tract symptoms (Novy 2008). The undetected chlamydia are able to ascend from the lower to the upper genital tract, evade the host's immune response and persist for long periods of time (McCormack 1979). The mechanisms leading to chlamydial persistence and subsequent damage to the fallopian tubes have only begun to be elucidated.

Pathogenesis of chlamydial infections in women

Research suggests that prior infection of the columnar epithelium of the cervix by chlamydia and/or Neisseria gonorrhoea is required before an ascending infection occurs. The majority ascend from the upper cervical os into the endometrial cavity and upwards to the fallopian tube and pelvic cavity (Course notes 2009).

However the precise mechanism that leads to the spread of chlamydia or other microorganisms from the lower to upper genital tract is poorly understood. Natural protection against ascending infection is provided by the mucous plug in the endocervical canal (Barrett 2005). Chlamydia may cause an endocervitis that damages the cervical canal and causes breakdown of the cervical plug (Barrett 2005). This may predispose to ascending infection (Barrett 2005).

Epidemiological studies suggest that sexual intercourse is necessary for the onset of PID (Barrett 2005, Course notes 2009). It is extremely rare in celibate women (Course notes 2009). It raises the question whether intercourse itself pushes bacteria into the upper genital tract. Bacteria may be carried by spermatozoa or by rhythmical contractions of the pelvic viscera during intercourse (Barrett 2005).

To explain the age related risk of chlamydia infection causing PID, it has been suggested that large areas of the columnar epithelium present on the cervix of young girls may predispose to ascending infection (Barrett 2005). There is also some evidence that the highest incidence of acute pelvic infection with chlamydia occurs just after menstruation (Barrett 2005). It may be that retrograde menstrual bleeding that occurs in the majority of menstruating women may facilitate the movement of organisms into the fallopian tubes (Barrett 2005).

Immunopathogenesis

Given the developmental cycle of chlamydia, there is strong evidence for the involvement of cell mediated immunity (Th-1 pathway) and its associated cytokines, interferon-y (IFN-y), interleukin-2 (IL-2) and andinterleukin-12 (IL-12), in resolving chlamydial infection (Debattista 2003). Tumour necrosis factor (TNF) may have a cytotoxic action and also a proinflammatory activity that promotes the production of other cytokines capable of blocking intracellular replication and may provoke cell death by apoptosis (Guerra-Infante 1999).

However response to chlamydial infection can result in a protective or pathological immune response. Being an intracellular pathogen, chlamydiae possess little intrinsic toxicity (Debattista 2003). Disease appears to result more from immune recognition to specific antigens expressed directly by the organism or indirectly on the surface of infected host cells (Ingalis 1995). Given the primary protective role affected by the cell mediated Th-1 response in resolving infection, inflammatory damage within the upper reproductive tract may be the result of a failed or weak Th-1 action resulting in chronic infection or as a result of an exaggerated or overstimulated Th-1 response (Debattista 2003).

A balance therefore exists between the protective and deleterious effects of cell mediated immunity (Debattista 2003). An effective immune response successfully clears infection, however a too weak or too strong cellular immune response could be responsible for pathology consequent to chronic chlamydial infection or hyper-inflammation respectively (Debattista 2003).

The model of pathogenesis by which chlamydia progresses from acute to chronic infection and finally serious disease (salpingitis, tubal occlusion) is yet to be fully determined. The pathways by which acute chlamydial infection achieves resolution or alternatively progresses to chronic infection with severe pathology appears to be varied and dependent on a closely entwined interplay between host and pathogen (Debattista 2003). A number of interconnected factors appear to play a significant role: human genetics (Khamesipour 1994), endocrinology (Crowley 2003), cytokine profile (Mosser 1999), previous infections (Debattista 2003), pathogen load (Pal 1998), chlamydial strain (Oritz 2000), the presence of other genital infections (Barrett 2005) and route of infection (Debattista 2003).

Immune pathways

An acute infection of chlamydial organisms requires colonisation of the intended site of infection (Debattista 2003). At this initial stage humoral immunity in the form of secretary IgA antibodies targeting the major outer membrane protein (MOMP) of the pathogen appears to be the significant protective response and the determinant of whether infection develops or is cleared (Su 1993). This protective immune response to infection is associated with serovar specific determinants on the MOMP (Allen 1991).

The MOMP is the principle surface protein component and is abundant in both infectious elementary bodies and intracellular reticular bodies (Debattista 2003). Functional MOMP probably serves as a porin, regulating the chlamydial developmental cycle by the passage of small molecules through the outer membrane (Stagg 1992). Antibodies to surface accessible epitopes on MOMP may neutralise infection and animal studies suggest a role for MOMP in protective immunity (Allen 1991).

Short lived immunity to chlamydia does develop after infection and is serovar specific as defined by serological variation in MOMP (Debattista 2003). The MOMP elicits chlamydia antibodies that recognise its surface exposed variable segments, therefore B-cell responses directed at serovar specific epitopes located in variable segments of MOMP may be critical to immunity (Debattista 2003). The MOMP contains serovar specific B-cell epitopes in four variable regions of the molecule whose amino acid sequence varies among serovars (Arno 1998).

After primary chlamydia infection immunity is considered to be only partially protective as the microorganism is sequestered within intracellular sites and it may be unrecognised by the immune system (Debattista 2003). Therefore this may predispose to its persistence in the genital tract and to recurrent infections (Brunham 1994). Evidence exists that the immune response is serospecific (Zhang 1989, Villeneuve 1994) which allows for reinfection with other chlamydial serovars or with the same serovar after antigenic mutations in the outer membrane protein (MOMP) (Morre 2000).

If organisms are not cleared by the initial humoral response, chlamydia will become established as intracellular parasites within the host's tissue (Debattista 2003). At this point antibody responses alone are inadequate to resolve what has now become an acute infection (Debattista 2003). The host response to this infection occurs within 1-2 days and is characterised by inflammation and mucosal infiltration with neutrophils, T-cell lymphocytes and monocytes (Barteneva 1996).

The B and T cells interacting synergistically identify chlamydial antigens on infected cell surfaces by specific antibody and subsequently lyse the infected cell by antibody dependent cellular cytotoxicity (Debattista 2003). Alternatively the production of specific antibody may inactivate free EBs preventing further infection (Romagnani 1996).

A possible pathway is the opposite of this overwhelming cellular immune response (i.e. a weak Th-1 response), leading to inadequate clearance of the infection (Debattista 2003). In this pathway the Th-2 response is dominant. A failure to mount a sufficient Th-1 cytotoxic T lymphocyte (CTL) response (Magee 1995), a premature shift from Th-1 to Th-2 pathways (Khamesipour 1994), production of higher levels of Th-2-inducing cytokines (Yang 1996), a defect in IL-2 (Mittal 1996), IL-12 or IFN-y secretion (Beatty 1994), or inadequate lymphoproliferation by T cells (Holland 1993) lead to defective activation of macrophages for bacterial killing and provide conditions for chlamydia to enter a chronic state (Debattista 2003).

Apoptosis induction occurs in human cells after infection with chlamydia (Debattista 2003). Intracellular chlamydial infection of macrophages can induce T-cell apoptosis through a secretory mechanism (Mgiga 2006). Apoptosis induction by chlamydia may explain how persistently infected macrophages escape T-cell surveillance and why T-cell responses are diminished during persistent chlamydia infection (Debattista 2003).

Experiments in vitro have established that IFN-y produced in response to the chlamydial infection blocks the intracellular life cycle of this organism and results in the formation of large aberrant reticulate bodies (Debattista 2003). However once IFN-y is removed, as would occur when an extracellular chlamydial infection is cleared, the aberrant forms revert to normal reticulate bodies and the typical chlamydial life cycle resumes (Beatty 1993). The reticulate bodies differentiate into elementary bodies, the infected cell lyses, and neighboring epithelial cells are infected (Beatty 1993). A similar intracellular chlamydial persistence may occur after treatment with some antibiotics (Beatty 1994). Each cycle of chlamydial growth and inhibition damages the fallopian tube epithelia by an immunologic mechanism, resulting in an increasing extent of fibrosis and eventual tubal occlusion (Witkin 1995).

Chlamydial heat shock protein 60 (cHSP60)

In an in vitro fallopian tube organ culture, C. trachomatis does not cause any visible damage (Cooper 1990). It has become increasingly evident that the immune response to a chlamydia infection, not the infection per se, induces fallopian tube occlusion (Land 2002). A single antigen, the HSP60 (expressed within 2-6 hours after infection), has been implicated in initiating a proinflammatory immune response after a chlamydia upper genital tract infection (Debattista 2003).

Under conditions of cell stress such as an increase in temperature or exposure to free oxygen or nitrogen radicals, HSP60 gene transcription greatly increases in an attempt to prevent protein denaturation and maximise cell survival (Debattista 2003). In an inactive but persistent chlamydial infection, synthesis of the major structural antigens ceases or is greatly reduced, however synthesis of HSP60 is increased (Beatty 1993). Microbial HSP60 is a potent inducer of proinflammatory cytokines (Beatty 1993).

A number of investigations have demonstrated a correlation between immunity to the cHSP60 and recurrent episodes of salpingitis, tubal occlusion and ectopic pregnancy (Wager 1990, Brunham 1992, Witkin 1994a). In women with a recent chlamydial cervical infection, immunity to chlamydial HSP60 is rarely observed (Witkin 1994a). This suggests that repeated infections or chlamydial persistence in the upper genital tract is needed for sufficient HSP60 to be released to initiate an immune response in the host (Novy 2008).

The homology (greater than 50%) between the chlamydial and human HSP60s also suggests that immune sensitisation to conserved HSP60 epitopes may result in autoimmunity to human HSP60 (Land 2002). Evidence of sensitisation to HSP60 epitopes shared between C. trachomatis and humans has been reported (Witkin 1994a, Yi 1993). In women sensitised to conserved HSP60 epitopes, expression of human HSP60 in the fallopian tubes (in response to cell damage or past infection by other microorganisms) reactivates HSP60 sensitised lymphocytes and induces an inflammatory response (Witkin 1994a). This may explain the sometimes puzzling observation of tubal inflammation in the apparent absence of infection (Debattista 2003).

Thus by allowing cHSP60 accumulation in the host, recurrence due to persistence and reinfections could be at the origin of harsh chlamydial disease (Mgiga 2006). Antibody responses to cHSP60 may signal chronic or persistent or repeated infection and may be indicative for chronic inflammatory tissue reaction.

Host susceptibility

Women susceptible to serious pathology may represent a subset of the population who are either more vulnerable to reinfection or to developing chronic infection through a predisposing lower capacity to mount a strong Th-1 response (Debattista 2003). Consequently they present a higher risk for pathological sequelae (Debattista 2003).

One study noted that a pre-existing antibody to cHSP60 in 280 female sex workers predicted a two to threefold increased risk for chlamydial PID and immune responses to cHSP60 antedate the chlamydial infection (Peeling 1997). There was also a link between cHSP60 antibodies and weaker responses to major outer membrane protein, a target for initial protective immunity. This may suggest that some women are genetically and immunologically selected towards pathology (Peeling 1997).

Genetic controls may determine the adequacy of Th-1 and Th-2 mechanisms at each stage of infection and pathogenesis by controlling for the:

* Level of cytokine secretion by infected mucosal epithelial cells

* Level of cytokine secretion by various lymphocyte cell subsets (i.e. cytokine receptivity)

* Overstimulation or understimulation of various negative and positive feedback mechanisms resulting in suppression or exaggerated enhancement (Debattista 2003)

Genetic control would similarly determine the sensitivity of recognition and cross reactivity of CD4 and CD8 T cells for a range cHSP60 epitopes as well as the strength of hypersensitivity response to these epitopes (Debattista 2003). Significant genetic variation across individuals would determine the relative immunogenicity and cross reactivity of significant epitopes responsible for lymphoproliferation (Debattista 2003).

It is possible for chlamydial infection to resolve spontaneously even in the absence of formal treatment (Norman 2002). It would appear that even in the absence of appropriate pharmacotherapy, at least 20-30% of women clear chlamydia infection spontaneously (Norman 2002). However spontaneous resolution of infection appears least likely in youngest women, in whom the prevalence is highest (Norman 2002).

Recognition of a peptide antigen by T lymphocytes requires binding of the antigen by major histocompatibility molecule (MHC molecules) (Ness 2004). MHC molecules are coded by a plethora of genes called HLA genes, with only a subset of the full range of HLA allelic variants carried by any given individual (Ness 2004). Not surprisingly HLA genotype is one of the most highly variant characteristics within populations, and HLA type plays a role in variability of the immune response (Ness 2004).

Previous studies, mostly conducted on sex workers in Nairobi, have associated chlamydia associated PID and tubal infertility (Ness 2004). One study demonstrated a link between HLA DQA (allelic variant) 0301, chlamydial cervicitis, endometritis and reduced fertility (Ness 2004). That HLA type could influence the genesis of PID and its consequences is biologically plausible (Ness 2004). It is accepted that recurrent chlamydial infection triggers a chronic inflammatory response. It is likely therefore that the more robust the predisposition to inflammation, the more robust the triggered response (Ness 2004).

Variations like single nucleotide polymorphisms (SNP) in immunological important host genes are thought to play a role in the course and outcome of chlamydia infection (den Hartog 2006). A recent study looked at whether genetic traits (carrying multiple SNPs in different genes) in the bacterial sensing system are associated with an aberrant immune response and subsequently with tubal pathology following a chlamydia infection (den Hartog 2006). The genes studied all encoded for pattern recognition receptors involved in sensing bacterial components. The authors found that carrying multiple single nucleotide polymorphisms in chlamydia PRR genes tends to result in an aberrant immune response and a higher risk of tubal pathology following infection (den Hartog 2006).

Further research is required.

Hysterosalpingo contrast sonography

Hysterosalpingo contrast sonography (usually shortened to HyCoSy) is a simple and well tolerated outpatient ultrasound procedure used to assess the patency of the fallopian tubes, as well as detect abnormalities of the uterus and endometrium (Course notes 2009). An echocontrast fluid is introduced into the uterine cavity via a 5 French cervical balloon catheter so that the uterine cavity, ovaries and fallopian tube patency can be assessed accurately. The use of transvaginal ultrasonography avoids exposure to x rays and is particularly suitable as a diagnostic test in patients with a low likelihood of tubal disease (Course notes 2009). Finding a normal cavity and bilateral fill and spill of contrast is reassuring, but where there is doubt hysterosalpingography or a laparoscopy and dye hydrotubation test should be done (Course notes 2009). Transvaginal ultrasonography can sometimes be useful in detecting hydrosalpinges (Course notes 2009).

Transvaginal hydrolaparoscopy (THL)

THL can be used to evaluate tubo ovarian pathology in the infertile woman (Course notes 2009). THL is less traumatic and is a more suitable outpatient procedure than standard laparoscopy (the risks of general anesthetic are avoided and there is less chance of trauma to major vessels) (Course notes 2009). Transvaginal access and systematic use of hydroflotation are potential advantages of THL for the exploration of tubo ovarian structures in infertility. Contraindicated in women with a retroverted uterus (to reduce risk of rectal perforation) (Course notes 2009).

Chlamydia and IVF

Evidence suggests that sensitisation to HSP60 may also interfere with reproductive success after IVF (Witkin 1994b, Witkin 1996). The early stage embryo (Bensuade 1983) and epithelial cells in the decidua (Mincheva-Nilsson 1994) express HSP60. A murine hybridoma specific for HSP60 was also shown to react with the surface of human and mouse trophoblasts (Heybourne 1994). HSP60 expression during pregnancy may reactivate HSP60 sensitised lymphocytes (Novy 2008). The resulting pro inflammatory immune response may directly interfere with embryo development or may disturb the balance of immune regulatory mechanisms needed to prevent rejection of the semiallogeneic embryo (Novy 2008).

Women undergoing IVF who had cervical IgA antibodies to chlamydial HSP60 had an increased rate of transient implantation after embryo transfer and a significantly poorer outcome than did antibody negative women (Witkin 1994b). Further analysis revealed that cervical immunity to a shared human HSP60 epitope and chlamydia was similarly correlated with IVF failure (Witkin 1996). Circulating systemic humoral immunity to human HSP60 has also been associated with a history of spontaneous abortion (Witkin 1996). An association between IVF failure, humoral immunity to chlamydia, and expression of human HSP60 in ovarian follicle fluid has been reported (Neuer 1997).

Herbal medicine and chlamydia Berberine

Berberine is an alkaloid present in Hydrastis canadensis, Coptis chinensis, Berberis aquifolium, Berberis vulgaris and Berberis aristata (Birdsall 1997, Berberine 2000). Amin et al (1969) screened a total of 54 microorganisms for sensitivity to berberine and found the alkaloid possess antimicrobial activity against gram positive and gram negative bacteria, fungi and protozoa.

Aqueous solutions of berberine have been employed in cases of ocular infections from C. trachomatis (Berberine 2000). A clinical study of aqueous berberine versus sulfacetamide for the treatment of chlamydia infection was conducted on 51 subjects in an outpatient eye clinic (Babbar 1982). It was determined that while sulfacetamide eye drops produced better clinical results, conjunctival scrapings of these patients remained positive for the infective agent and relapses occurred. In contrast the conjunctival scrapings of patients receiving the berberine chloride eyedrops were negative for chlamydia and there were no relapses, even one year after treatment (Babbar 1982). It was concluded that while berberine chloride had no direct antichlamydial properties, it seemed to be that the antitrachoma effects were related to protective host mediated factors (Babbar 1982).

A second clinical study found berberine chloride superior to sulfacetamide in both clinical course of trachoma and achieving a drop in serum antibody titers against chlamydia (Khosla 2000).

A series of in vitro and in vivo experiments were conducted using chick embryos to further determine if the antitrachoma effects were related to direct antichlamydial properties or to host mediated factors (Babbar 1982). Three different chlamydia isolates were incubated in vitro with 0.2% berberine chloride prior to inoculation into chick embryos. A different set of embryos was initially infected with the chlamydia isolates and then subsequently given three doses of 0.2% berberine chloride. The in vitro incubation of chlamydia with berberine had no effect in reducing the lesion scores or the mortality of the organism, whereas the administration of the berberine to infected embryos resulted in the elimination of both lesion and mortality. The authors suggested that with chlamydia, berberines method of action is by stimulating some protective mechanism in the host (Babbar 1982).

Polyherbal formulations

Topical microbicides have been suggested to assist control chlamydial infections. Polyherbal formulations that have been studied include BASANT and Praneem.

BASANT antichlamydial topical microbicide: Aloe vera (2.5%w/v), purified extracts of Emblica officinalis (amla) 2.5% w/v), diferuloylmethane (curcumin) (0.36% w/v), purified saponins from Sapindus mukorossi (1% w/v), and rose water (Bengraj 2008).

BASANT is said to have a synergistic effect inhibiting chlamydia infection (Bengraj 2008). In vitro infectious EBs of chlamydia were decreased as seen by inclusion counts with respect to the time of incubation and the concentration of BASANT. Bhenraj et al (2008) suggest BASANT has the potential for clinical use. Intravaginal application of BASANT prior to sexual contact may kill the infectious EBs before they infect the target epithelial cells, thus preventing chlamydial infection (Bengraj 2008). The mechanism by which BASANT inhibits chlamydia is not known and requires further investigation.

BASANT was observed to have no cytotoxic effect on the epithelial adenocarcinoma cells, which are the primary target of various pathogens (Bengraj 2008). Talwar et al (2008) showed it did not cause any abnormalities in the structural integrity of vaginal epithelium in vivo and did not have any systemic effect as evidenced by blood chemistry and hematology after 7 consecutive days use of twice daily application for 3 weeks in the vagina.

Two formulations of Praneem have been studied (Garg 2002):

1. Praneem polyherbal pessary (PR-048): Azadirachta indica purified leaf extract, Sapindus mukerossi purified saponins, Mentha citrata oil.

2. Praneem polyherbal cream (CH-005): Sapindus mukerossi purified saponins, Mentha citrata oil and a natural polycationic polymer.

Both formulations were effective in blocking vaginal transmission of chlamydia in a mouse model. In a clinical study, Garg et al (2002) reported anti chlamydial activity. Out of 28 patients with chlamydial cervicitis, 22 patients recovered clinically and microbiologically after 7-21 days of application of Praneem cream.

The authors suggested a safe spermicidal formulation with wide spectrum antimicrobial action would be a desirable addition to the presently available spermicides (Garg 2002).

Immunolodulatory

Echinacea spp.

Immunological changes associated with Echinacea radix can be categorised as non specific (i.e. directed against a broad range of microorganisms, e.g. neutrophils that phagocytose and kill bacteria) and/or specific (i.e. directed against a single organism, e.g., antibody response) (Rasmussen 2009). Non specific immune responses associated with Echinacea include increased numbers of circulating white blood cells, granulocyte migration, monocytes, neutrophils and natural killer cells, and the phagocytic abilities of these (Rasmussen 2009). Although this would indicate an immunostimulant pharmacological action of Echinacea, traditional use and research also provides evidence of immunomodulatory and anti inflammatory actions (Rasmussen 2009, Chica 2009).

Preclinical studies indicate that Echinacea constituents modulate immune mechanisms and there is increasing evidence that lipophilic Echinacea preparations containing N-alkylamides can suppress stress related cellular immune responses (Gertsch 2004, 2008, Raduner 2006, Mattias 2007, Sasagawa 2006). Gertsch et al (Gertsch) analysed the standardised tincture Echinaforce[R] and found that it induced de novo synthesis of tumor necrosis factor alpha (TNF-[alpha]lpha) mRNA in primary human monocytes/macrophages, but not TNF-[alpha]lpha protein. Interaction of N-alkylamides with the human cannabinoid receptor type-2 (which is a modulator of inflammation) may provide a mechanistic basis for anti inflammatory and immunomodulatory effects exerted by Echinacea.

Chicca et al (2009) demonstrated that N-alkylamides act in concert and exert pleiotropic effects modulating the endocannabinoid system by simultaneously targeting the CB2 receptor, endocannabioid transport and degradation. The expression of anti-inflammatory cytokine IL-10 was significantly superstimulated while the expression of the pro-inflammatory protein TNF-alpha was inhibited (Chicca 2009). IL-10 has profound effects on activated macrophages and antigen-presenting cells (Florentino 1991). Extensive genetic and pharmacological studies have established that IL-10 plays an essential, non redundant role in limiting chronic and acute inflammation (Florentino 1991).

Specific Echinacea alkylamides have also been identified that inhibit both 5-lipoxygenase and cyclooxygenase, upstream enzymes involved in the production of leukotrienes and prostaglandins (Muller-Jakie 1994, Clifford 2002).

IL-2 is a critical autocrine growth factor upregulated upon T cell activation that is required for the clonal expansion of T cells (Gillis 1980). IL-2 is also an important cytokine involved in regulatory T-cell activation and expansion (Antony 2005). Thus modulatory effects on IL-2 protein production may influence specific immune responses to infection (Sasagawa 2006). In vivo, 4 weeks of oral administration of Echinacea aerial parts increased serum IL-2 levels 320-fold in rats (Mattias 2007). In vitro, IL-2 mRNA production was decreased over a 24 hour period in human peripheral blood leukocytes treated with an Echinacea purpurea fresh plant extract (Gertsch 2008).

Alkylamides in an E. purpurea preparation were found in vitro to have an inhibitory effect on IL-2 production but not cell viability in a dose dependent manner (Sasagawa 2006). The alkylamides suppressed the ability of activated Jurkat T cells to produce IL-2 independently of direct, cytotoxic effects (Sasagawa 2006).

Preliminary studies on the regulation of human gene expression by a commercially blended Echinacea product (Echinaforce[R]) demonstrated that the overall gene expression at 48 hours to 12 days after taking Echinacea was consistent with an anti inflammatory response (Randolph 2003). The expression in IL-1[beta], TNF-[alpha], intracellular adhesion molecule and IL-8 was modestly decreased up to day 5, returning to baseline by day 12 (Randolph 2003).

The antigen specific in vivo immunomodulatory potential of continuous treatment with Echinacea and goldenseal (Hydrastic canadensis) root extract was investigated over a period of 6 weeks using rats that were injected with the novel antigen keyhole limpet hemocyanin (KLH) and re-exposed to KLH after the initial exposure (Rehman 1999). Immunoglobulin production was monitored via ELISA continuously over a period of 6 weeks. The Echinacea treated group showed a significant augmentation of their primary and secondary IgG response to the antigen, whereas the goldenseal treated group showed an increase in the acute primary IgM response during the first 2 weeks of treatment. The authors suggested that Echinacea or goldenseal may enhance immune function by increasing antigen specific immunoglobulin production (Rehman 1999).

Hydrastis canadensis (goldenseal)

Specific indications in traditional western herbal medicine for goldenseal include its use for subacute or chronic inflammation of the mucous membranes, particularly when accompanied by discharge or catarrh (Felter 1905). Kings American Dispensary also describes goldenseal as having a beneficial action in the cure of fungoid endometritis, lacerated cervix, and pelvic cellulitis. Locally and internally excellent results are obtained from Hydrastis in leucorrhoea, both vaginal and uterine. For gonorrhoea, Lloyd's Hydrastis probably enjoys a more extensive use as a local application than any other drug, and this use of it is not confined to Eclectic practitioners alone (Felter 1905). Therefore goldenseal could be indicated as a pessary to assist repair of mucous membranes of the vagina and cervix from chlamydial infection and to also perhaps act as a local bactericide (due to berberine content).

Picrorrhiza kurroa (picrorrhiza)

Picrorrhiza is traditionally indicated for poor immunity and recurrent infections (Mills 2000). A recent study was performed on the biopolymeric fraction RLJ-NE-205 that was isolated and purified from the rhizomes of P. kurroa. The effects of RLJ-NE-205 were evaluated on the in vivo immune function of the mouse (Gupta 2006). At the dose of 50 mg/kg, significant increases in the proliferation of lymphocytes and cytokine levels (IL-4 and IFN-gamma) in serum were observed. A dose dependent increase was demonstrated in hemagglutination antibody titre, delayed type hypersensitivity, phagocytic index and CD4/CD8 population. This suggests that the biopolymeric fraction RLJ-NE-205 improves the immune system and might be regarded as a biological response modifier (Gupta 2006).

Baicalin

In order to survive and successfully replicate, chlamydia have evolved a number of strategies to cope with the harsh intracellular environment, such as avoiding fusion with lysosomes, prevention of host cell apoptosis and altering cell signal transduction (Debattista 2003). Baicalin is a flavonoid isolated from Scutellaria baicalensis and is known to have an effect on multiple biological functions, including antimicrobial activity (Mills 2000). Recently a chlamydia secreted protein, chlamydia protease like activity factor (CPAF), has been identified (Zhong 2001). CPAF is responsible for degrading host transcription factors such as Regulatory Factor X5 (RFX5) required for major histocompatibility complex antigen expression, which may allow chlamydia to escape efficient immune detection (Fan 2002). It is evident that CPAF play a critical role in inflammation induced by chlamydia (Stephens 2003).

Huang et al (2009) demonstrated baicalin treatment reduced expression of CPAF in the hep-2 cells infected by Chlamydia serovars D. They also detected that baicalin increased expression of RXF5 mRNA and protein. They proposed that CPAF is a primary target of baicalin and may play an important role in down regulating RXF5 and showed the inhibition of chlamydia infection in the cell lines treated by the baicalin. The authors concluded that the data allowed them to draw that baicalin is an effective anti chlamydial agent which could, to some extent, down regulate the expression of CPAF resulted from inhibition of chlamydia growth and could represent a powerful investigative and potentially therapeutic strategy in the prevention and treatment of infectious disease caused by chlamydia infection (Hyang 2008, 2010).

Centella asiatica (gotu kola)

As chlamydial PID is associated with tissue damage and adhesions, gotu kola could be indicated. Gotu kola has several pharmacological actions: for example after oral and topical administration in rats, increased cellular hyperplasia and collagen production were noted at the site of injury, measured by increased granulation tissue levels of DNA, protein, collagen and hexosamine (Centella 2007). More rapid maturation and cross linking of collagen were seen in animals treated with the herbal extract, as evidenced by elevated stability of acid soluble collagen and increases in aldehyde content and tensile strength (Suguna 1996).

Asiaticoside, a constituent isolated from Centella asiatica, promotes fibroblast proliferation and extracellular matrix synthesis in wound healing (Lu 2004). The precise mechanism however, in molecular and gene expression levels, still remains partially understood. Using cDNA microarray technology the alternation of genes expression profiles was determined in a human dermal fibroblast in vitro in the presence of asiaticoside. Fifty four genes with known functions for cell proliferation, cell cycle progression and synthesis of the extracellular matrix, were significantly up regulated in our 'whole genes nest' expression profile at various time points. Furthermore mRNA levels and protein productions of certain genes responsible for extracellular matrix (ECM) synthesis (e.g. encoding type I and type III collagen proteins) were evaluated by Northern blot and radioimmunoassay respectively. As a result there is a close correlation among the gene profile, mRNA and protein production in the cells response to asiaticoside stimulation (Lu 2004).

Asiaticoside activity has also been studied in normal and delayed wound healing (Shukla 1999). Asiaticoside was active by the oral route also at 1 mg/kg dose in a guinea pig punch wound model. It promoted angiogenesis in the chick chorioallantoic membrane model at 40 [micro]g/ disk concentration. Oral administration in normal animals significantly enhanced the rate of wound healing as assessed by increase in collagen synthesis and tensile strength of the wound tissues (Shukla 1999). These results indicate that asiaticoside exhibits significant wound healing activity in normal as well as delayed healing models and is the main active constituent of gotu kola (Shukla 1999).

Gotu kola has long been recommended for the treatment of keloids and/or hypertrophic scars (Centella 2007). In one open clinical trial 227 patients were divided into two groups and treated with oral gotu kola alone (139/227) or surgical scar revision plus gotu kola at doses of 60-150 mg daily for up to 18 months. In the gotu kola only group 82% (116/139) experienced relief of symptoms and disappearance of inflammation. In the combined surgery and gotu kola group 72% (63/88) demonstrated improvement (Bosse 2007).

Future

The best prevention of chlamydia is to detect and treat early stage asymptomatic and symptomatic infections. This can be achieved by the screening of all sexually active reproductive age women and by educating clinicians and patients on the importance of this testing. An Australian study determined that combining chlamydia and Pap screening increases the rate of chlamydia screening in general practice, and that implementing this approach would require little additional infrastructure support in settings where a cervical screening program already exists (Bowden 2008).

A clearer picture of the natural history of chlamydial pathology may assist in providing better predictors of those women who may go on the develop significant sequelae after infection and knowledge of the pathogenesis and possible host susceptibility could assist in predicting those who may develop serious disease, including infertility, and may contribute to improved management of such persons during earlier stages of infection and assist in prevention.

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Nadine Campbell

ashaholistichealth@bigpond.com

Nadine Campbell is a naturopath and herbalist who runs Asha Holistic Health in Wollongong and Sydney. Nadine also practices at the community based non profit Illawarra Women's Health Centre. She holds a degree in Biomedical Science from the University of Wollongong and graduated with Distinction from the University Western Sydney (UWS) with both a Degree in Naturopathy and a Postgraduate Diploma in Naturopathy, as well as being awarded the UWS Deans Medal for outstanding academic performance. She is currently studying her Masters Degree in Reproductive Medicine at University of New South Wales and is particularly passionate about women's health and fertility issues.
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